&#34;Methods and Systems for Achieving a Physiological Response by Pudendal Nerve Stimulation and Blockade&#34;

ABSTRACT

Methods and apparatus are therefore provided herein for stimulating a desired physiological effect. The methods and apparatus can be used to control micturition, defecation and/or ejaculation. The methods and apparatus also can be used to control pain in the lower pelvic region, for example and without limitation, interstitial cystitis. The methods and apparatus also can be used to increase sexual sensation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 14/276,297, filed May 13, 2014, which is a divisionalapplication of U.S. patent application Ser. No. 12/184,518, filed Aug.1, 2008, which claims the benefit under 35 U.S.C. §119(e) to UnitedStates Provisional Patent Application Nos. 60/953,502, filed Aug. 2,2007, and to U.S. Provisional Patent Application No. 60/955,212, filedAug. 10, 2007, each of which are incorporated herein by reference in itsentirety.

Described herein are methods for achieving a physiological response,such as micturition, defecation, ejaculation and pain relief bystimulating and/or blocking a pudendal nerve or a branch thereof.

A number of conditions arise from disruption of normal physiologicalprocesses in the lower pelvis. Conditions, such as urinary incontinence,overactive bladder, urine retention and voiding dysfunction, detrusorsphincter dyssinergia, fecal incontinence, constipation, irritable bowelsyndrome, sexual dysfunction in both men and women, prematureejaculation, decreased sexual sensation, an orgasm, urethral pain,prostate pain, vulvodynia, anal pain, rectal pain and bladder pain areamong those conditions. Those conditions can result from neurologicalimpairment or from other diseases or conditions. For example, urinaryincontinence can result from spinal cord injury or stroke, or damagecaused by trauma, disease (e.g., multiple sclerosis) and/or congenitaldefects.

The pudendal nerve originates in the sacral plexus. It derives itsfibers from the ventral branches of the second, third, and fourth sacralnerves (S2, S3, S4). It passes between the piriformis and coccygeusmuscles and leaves the pelvis through the lower part of the greatersciatic foramen. It then crosses the spine of the ischium, and reentersthe pelvis through the lesser sciatic foramen. It accompanies theinternal pudendal vessels upward and forward along the lateral wall ofthe ischiorectal fossa, and is contained in a sheath of the obturatorfascia termed the pudendal canal. The pudendal nerve gives off theinferior rectal nerves. It soon divides into two terminal branches: theperineal nerve, and the dorsal nerve of the penis (males) or the dorsalnerve of the clitoris (in females). The inferior anal nerves branch offshortly after passing through the greater sciatic foramen. The perinealnerve is the more superficial terminal branch of the pudendal nervewhile the dorsal nerve of the penis or dorsal nerve of the clitoris aredeeper terminal branches of the pudendal nerve, traveling into the deepperineal pouch (see, e.g., U.S. Pat. No. 7,047,078, FIGS. 1 and 2, andaccompanying description for a useful diagram).

The pudendal nerve carries both sensory (afferent) and motor (efferent)signals. It innervates, among other things, the anal and externalurethral sphincters. It also innervates the penis and clitoris,bulbospongiosus and ischiocavernosus muscles, and areas around thescrotum, perineum, and anus. At sexual climax, peristaltic action ofmuscles in the reproductive ducts and accessory glands (e.g., seminalvesicles, prostate and Cowper's (bulbourethral) glands, along withspasms in the bulbospongiosus and ischiocavernous muscles result inejaculation in the male. Spasms in the bulbospongiosus andischiocavernous muscles accompany most of the feelings of orgasm in bothsexes.

Micturition, also called voiding or urination, is the act of emptyingthe bladder. In humans, when about 200 ml of urine has accumulated,distension of the bladder wall typically activates stretch receptors,triggering a visceral reflex arc. Afferent impulses are transmitted tothe sacral region of the spinal cord, and efferent impulses return tothe bladder via the parasympathetic pelvic nerves, causing the detrusormuscle of the bladder to contract and the internal sphincter of thebladder to relax. As the contractions increase in intensity, they forcestored urine through the internal sphincter into the upper part of theurethra. Afferent impulses are also transmitted to the brain, so onefeels the urge to void at this point. Because the external urethral(urinary) sphincter is voluntarily controlled, a person can choose tokeep it closed and postpone bladder emptying temporarily. On the otherhand, if the time is convenient, the voluntary sphincter can be relaxed,allowing urine to be expelled from the bladder. When one chooses not tovoid, reflex bladder contractions subside within a minute or so andurine continues to accumulate. After 200-300 ml more has collected, themicturition reflex occurs again and, if urination is delayed again, isdamped once more.

Thus, normal bladder activity is typically divided into two phases. Inthe first phase, the “storage phase,” the bladder detrusor is quiet andthe EUS is closed. In the second phase, the “voiding phase,” the bladderdetrusor contracts and the EUS is (voluntarily) relaxed, permittingurine to flow out of the urethra. In patients with neurological damageaffecting the micturition process, this process is disrupted, leadingto, for example, incontinence or retention.

Incontinence is the inability to control micturition. Incontinencetypically is a result of emotional problems, physical pressure duringpregnancy, or nervous system problems, such as stroke or spinal cordlesions.

In urinary retention, the bladder is unable to expel its containedurine. Urinary retention is common after general anesthesia has beengiven (it seems that it takes a little time for the smooth muscles toregain their activity). Urinary retention in men often reflects prostatehypertrophy, narrowing the urethra, making it difficult to void.Stretching of the bladder wall by urine causes sensory impulses to betransmitted to the sacral region of the spinal cord. Motor impulses aredelivered to the bladder detrusor muscle and the internal sphincter viaparasympathetic fibers of the pelvic nerves. The pudendal nerve servesthe striated muscle fibers of the external urethral sphincter.

Defecation proceeds by a similar manner as micturition. Sensory andmotor control of defecation travels through the pudendal nerve. Therectum usually is empty. When feces are forced into the rectum by massmovement, the rectal wall is stretched, initiating the defecationreflex. In the defecation reflex, the walls of the sigmoid colon andrectum contracts and the anal sphincters relax, forcing the feces intothe anal canal. The brain, however, decides whether the passage of fecesshould be temporarily stopped. If they are stopped, the rectal wallsrelax, until another mass-movement initiates another defecation reflex.

Patients with supra-sacral spinal cord injuries typically have novoluntary control over the micturition, defecation and ejaculatoryprocesses. For example, after spinal cord injury (SCI) incontinenceoccurs frequently due to detrusor overactivity. Meanwhile, the bladderalso does not empty well due to detrusor sphincter dyssynergia (DSD)resulting in a large residual volume of urine. Thus, the management ofbladder function after SCI is a challenging task, because it requiresinhibition of detrusor overactivity during urine storage and inductionof a large amplitude bladder contraction to empty the bladder (Boggs JW, Wenzel B J, Gustafson K J, Grill W M. Spinal micturition reflexmediated by afferents in the deep perineal nerve. J Neurophysiol 93:2688-2697, 2005). Current treatment for bladder dysfunction after SCIhas either limited success (Yoshimura N, Smith C P, Chancellor M B, deGroat W C. Pharmacologic and potential biologic interventions to restorebladder function after spinal cord injury. Cur Opin Neurol 13: 677-681,2000) or requires major invasive spinal surgery to implant stimulatingelectrodes on spinal roots (Brindley G S. The first 500 sacral anteriorroot stimulator implants: general description. Paraplegia 32: 795-805,1994). Intermittent urethral catheterization is the most common methodfor managing urinary tract dysfunction. However, it can lead to frequentbladder infections.

U.S. Pat. No. 7,047,078 B2 and related United States Patent PublicationNo. 2006/0184208 A1, describes methods and apparatus for stimulatingcomponents in, on, or near the pudendal nerve or its branches in orderto elicit a physiological response. Those documents describe the use ofelectrodes to directly stimulate or depress bladder contractions. Bythis method the depression of detrusor activity during the storagephase, and detrusor contraction can be achieved during a “voidingphase.” Nevertheless, this method has been found to be unsatisfactory inachieving complete voiding, because it can not completely relax the EUS.

In another method, implemented by, e.g., a FineTech-Brindley (VOCARE)stimulator, micrutition and/or defecation is accomplished by pulsatilestimulation of the sacral spinal anterior roots. Brindley's methodrequires a major spinal surgery to open the spinal bone and cut thesacral spinal posterior roots, which eliminates the spinal reflexes fordefecation and sexual functions. The electrical pulses typicallygenerated by this device causes contraction of both the bladder detrusorand the EUS. Because the bladder detrusor comprises smooth muscle, whilethe EUS comprises striated muscle, the EUS releases before the detrusor.During the short time period between EUS release and detrusor release,voiding can occur. The pulses are repeatedly applied until the bladderis emptied.

The Medtronics InterStim® device may be used to treat detrusorhyperreflexia (DH, also called detrusor overactivity) or overactivebladder. It does not induce bladder excitation or urination (voiding).The InterStim® device sends electrical pulses to the sacral nerve toinfluence the bladder and surrounding muscles that manage urinaryfunction.

In Seif, Ch., et al. (Selective Block of Urethral Sphincter ContractionUsing a Modifed Brindley Electrode in Sacral Anterior Root Stimulationof the Dog, Neurourology and Urodynamics 21:502-510 (2002)), anelectrode of a FineTech-Brindley type device was used to stimulate, bylow frequency (e.g., 20 Hz) an anterior S2 root in order to preventtransmission of motor impulses to the EUS in dogs. Other than arhizotomy of all posterior roots from S1 to S3, the dogs were healthy,including having intact spinal cords. Although occasionally effective topermit voiding, it does not address, or prevent the concomitant EUScontraction and detrusor contraction present in for example, DSD, orstimulated by the activation of the detrusor by the device.

This selective stimulation method activates the small nerve in thesacral spinal root without activating the large nerve. The “block”described in In Seif, Ch., et al. was not a blockage of, e.g., theconduction of the pudendal nerve, rather it did not activate them infact. As a result, the detrusor-sphincter-dyssynergia (DSD) occurringafter spinal cord injury will still cause EUS contraction when thebladder contracts. Because, In Seif, Ch., et al. used dogs with anintact spinal cord, there was no DSD in this animal model. It isbelieved that the methods described in In Seif, Ch., et al., would notprovide any better results in DSD patients using the FineTech-Brindleydevice.

Further, in Seif, Ch., et al., the method requires opening up of thevertebrae and cutting the spinal sensory roots. This further damages thenerve systems and results in loss of reflex defecation, reflex penileerection, reflex ejaculation, etc. There have been studies showing thatthe method described in Seif, Ch., et al., do not work well if thesensory spinal roots are not cut (Kirkham, A. P. S., Knight, S. L.,Craggs, M. D., Casey, A. T. M. and Shah, P. J. R., Neuromodulationthrough sacral nerve roots 2 to 4 with a Finetech-Brindley sacralposterior and anterior root stimulator. Spinal Cord, 40:272-281, 2002.and Van Kerrebroeck, P. E. V., Koldewijn, E. L., Rosier, P. F. W. M.,Wijkstra, H. and Debruyne, F. M. J., Results of the treatment ofneurogenic bladder dysfunction in spinal cord injury by sacral posteriorroot rhizotomy and anterior sacral root stimulation. J. Urol.,155:1378-1381, 1996).

Thus, there is a need for methods and apparatus that can effectivelycontrol the micturition, defecation, pelvic pain and/or sexual response

STATEMENT REGARDING FEDERAL FUNDING

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Grant No.1RO1-DK068566-01, awarded by the National Institutes of Health.

SUMMARY

Methods and apparatus are therefore provided herein for stimulating adesired physiological effect. The methods and apparatus can be used tocontrol micturition, defecation and/or ejaculation. The methods andapparatus also can be used to control pain in the lower pelvic region,for example and without limitation, interstitial cystitis. The methodsand apparatus also can be used to increase sexual sensation.

In one non-limiting embodiment, the pudendal nerve, or a branch thereof,is stimulated by electric pulses in a frequency range of from 0.5 to 15Hz to inhibit bladder or rectal contractions and to reduce pelvic painof bladder, urethra, prostate, anus, or rectum, such as frominterstitial cystitis. In another non-limiting embodiment, the pudendalnerve, or a branch thereof, is stimulated by electric pulses in afrequency range of from 15 to 50 Hz to elicit bladder contractions,rectal contractions, ejaculation, orgasm and/or sexual arousal. Theelectrical pulses in a frequency range of from 15 to 50 Hz may beapplied intermittently, such as for two or more stimulation intervals of0.5 to 60 seconds, with a suitable time period between the intervals,such as from 0.5 seconds to 5 minutes, to facilitate and/or optimizemicturition, defecation, ejaculation, orgasm or sexual stimulation.

In the case of micturition and defecation, the pudendal nerve, distal tothe point of stimulation by electrical pulses in a frequency range offrom 15 to 50 Hz, may be co-stimulated with electrical pulses in afrequency range of greater than 4 kHz, for example and withoutlimitation, from 4 kHz to 50 kHz, to block activity of the EUS and/oranal sphincter, thereby facilitating micturition and defecation.

The electrical pulses may be applied by an implanted device, withimplanted electrodes stimulating a pudendal nerve or a branch thereof,or to the skin (transdermally, intradermally or subdermally) to asuperficial branch of the pudendal nerve, typically located perianallyor perigenitally. Systems and devices are described herein forimplementing the described methods. In non-limiting embodiments, thesystems comprise a pulse generator and a controller for generating andcontrolling parameters of the electrical pulses. The pulse generator maybe implanted in a subject or used externally in the case of applyingelectric pulses to the skin. Additional non-limiting embodiments anddetails and variations to these embodiments are provided herein and inthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of one non-limiting embodiment of a deviceuseful in implementing methods described herein.

FIG. 2. (A) Experimental setup. (B) Pudendal nerve stimulation (20 Hz)and block (10 kHz) delivered to each electrode. The 20 Hz stimulation atelectrode #1 activates an excitatory pudendal-to-bladder spinal reflexto induce bladder contraction, while the 10 kHz stimulations atelectrodes #2 and #3 block pudendal nerve conduction bilaterally toprevent EUS contraction. EUS=external urethral sphincter.

FIG. 3. Voiding reflex induced by bladder distension in a normal (A) anda chronic SCI (B) cat. (A) In a normal cat, the bladder was infused at0.5 ml/min. At the infusion stop a total of 21 ml was infused, and 20 mlwas voided with a voiding efficiency of 95.2%. (B) In a chronic SCI cat(11 months after SCI), the bladder was infused at 4 ml/min. At theinfusion stop a total of 74 ml was infused, but only 4 ml was voidedwith a voiding efficiency of 5.4%.

FIG. 4A-4B. Maximal bladder pressure (A) and average flow rate (B)during voiding in spinal intact and chronic SCI cats (N=3). Spinalintact—during voiding induced by bladder distension in spinal intactcats (see FIG. 3A). Control—during voiding induced by bladder distensionin chronic SCI cats (see FIG. 3B). 20 Hz±block 2—during voiding inducedby 20 Hz stimulation of the left pudendal nerve with 10 kHz blocking ofboth left and right pudendal nerves in chronic SCI cat (see FIG. 8B).*Indicates statistical significance (P<0.05).

FIG. 5. Voiding efficiency in spinal intact and chronic SCI cats (N=3).Spinal intact—efficiency of voiding induced by bladder distension inspinal intact cats (see FIG. 3A). Control—efficiency of voiding inducedby bladder distension in chronic SCI cats (see FIG. 3B). 20 Hz±block1—efficiency of voiding induced by 20 Hz stimulation and 10 kHz blockingof the left pudendal nerve in chronic SCI cats (see FIG. 8A). 20Hz±block 2—efficiency of voiding induced by 20 Hz stimulation of theleft pudendal nerve with 10 kHz blocking of both left and right pudendalnerves in chronic SCI cat (see FIG. 8B). * Indicates statisticalsignificance (P<0.05).

FIG. 6. Voiding reflex induced by bladder distension in chronic SCIcats. (A) In a chronic SCI cat (9 months after SCI), the bladder wascontinuously infused at 3 ml/min. At the first voiding contraction, atotal of 20 ml was infused into the bladder. At the last voidingcontraction, a total of 57 ml was infused, but a total of 38 ml wasvoided by a series of short-lasting bladder contractions resulting in 19ml residual volume in the bladder. (B) In the same chronic SCI cat asshown in FIG. 3B, the bladder capacity was increased to 124 ml at theinfusion stop by the inhibitory 3 Hz pudendal nerve stimulation. Theblack bar under the bladder pressure trace indicates the duration of thepudendal nerve stimulation. After stopping the 4 ml/min infusion and thestimulation, several short-lasting bladder contractions voided a totalof 31 ml.

FIG. 7. Voiding reflex induced by 20 Hz pudendal nerve stimulation alonein a chronic SCI cat (9 months after SCI). The 10 kHz blockingstimulation was only briefly applied bilaterally to the pudendal nervesduring the 20 Hz stimulation of the left pudendal nerve. Twenty-fourmilliliter was infused into the bladder when the infusion was stopped.Twelve milliliter was voided only during the blocking stimulation. Theblack bars on bladder pressure trace indicate the stimulation durations.Infusion rate: 2 ml/min.

FIG. 8. Voiding reflex induced by stimulating and blocking the pudendalnerves in chronic SCI cats. (A) Both 20 Hz and 10 kHz stimulations wereapplied to the left pudendal nerve in a chronic SCI cat (11 month afterSCI). Eighty-six milliliter was infused into the bladder when theinfusion was stopped. A total of 53 ml was voided with a voidingefficiency of 61.6%. Infusion rate: 4 ml/min. (B) The 20 Hz stimulationwas applied to the left pudendal nerve during 10 kHz bilateral blockingstimulation of the pudendal nerves in a chronic SCI cat (9 month afterSCI). Twenty-five milliliter was infused into the bladder when theinfusion was stopped. Twenty-two milliliter was voiding during the 20 Hzstimulation with a voiding efficiency of 88%. Infusion rate: 2 ml/min.The black bars on bladder pressure traces indicate the stimulationdurations.

FIG. 9. Schematic drawing of the electrode attachment described inExample 2.

FIG. 10A-10D. Frequency-dependent inhibitory effect on rhythmic bladderactivity induced by electrical perigenital stimulation. A: effect onbladder pressure traces at different stimulation frequencies.

The black bars under bladder pressure traces mark the stimulationduration. B: area under bladder pressure curve. C: reciprocal ofintercontraction interval (1/ICI). D: average bladder contractionamplitude. Bladder responses during stimulation were normalized to theresponse before stimulation in B-D. Stimulation: 30 V in A, 5-30 V inB-D; 0.2-ms pulse width. The calibration bars in A apply to all bladderpressure recordings. *Statistical significance (P<0.05); n=3.

FIG. 11A-11D. Intensity-dependent inhibitory effect on rhythmic bladderactivity induced by electrical perigenital stimulation. A: effect onbladder pressure traces at different intensities. The black bars underbladder pressure traces mark the stimulation duration. B: area underbladder pressure curve. C: reciprocal of ICI (1/ICI). D: average bladdercontraction amplitude. Bladder response during stimulation wasnormalized to the response before stimulation in B-D. Stimulation: 7 Hz;0.2-ms pulse width. The calibration bars in A apply to all bladderpressure recordings. *Statistical significance (P<0.05); n=3.

FIG. 12A-12D. Bladder contractions induced by electrical perigenitalstimulation at different frequencies (A) or at different intensities(B). The area under bladder pressure curve is dependent on bothstimulation frequency (C) and intensity (D). Stimulation: 15 V in A,15-30 V in C; 30 Hz in B and D; 0.2-ms pulse width. The black bars underbladder pressure traces mark the stimulation duration. The response wasnormalized to the maximal response during each trial in C-D. Thecalibration bars in A apply to bladder pressure recordings in both A andB. *Statistical significance (P<0.05); n=4.

FIG. 13A-13D. Inhibitory effect of electrical perigenital stimulation onbladder activity during cystometrograms (CMGs). A: bladder pressuretraces. The initial empty bladder was infused with saline at 2 ml/min.Control: no stimulation. Stimulation: 7 Hz; 20 V; 0.2-ms pulse width.The black bar under bladder pressure trace marks the stimulationduration. B: bladder capacity was significantly increased by electricalperigenital stimulation at 7 Hz. C: volume threshold to induce the firstpremicturition contraction (PMC) was also significantly increased. D andE: PMC amplitude and number of PMCs per minute were not changed.Responses in B-E were normalized to the measurements during firstcontrol CMG. Stimulation in B-E: 5-30 V; 0.2-ms pulse width. Thecalibration bars in A apply to all bladder pressure recordings.*Statistical significance (P<0.05); n=4.

FIG. 14. Excitatory effect of electrical perigenital stimulation onbladder activity during CMGs. A: bladder pressure traces. The initialempty bladder was infused with saline at 2 ml/min. Control: nostimulation. Stimulation: 30 Hz, 15 V, 0.2-ms pulse width. The black barunder bladder pressure trace marks the stimulation duration. B: bladdercapacity was significantly reduced by electrical perigenital stimulationat 30 Hz. Bladder capacity was normalized to the capacity measured inthe first control CMG. Stimulation: 10-30 V; 0.2-ms pulse width. Thecalibration bars in A apply to all bladder pressure recordings.*Statistical significance (P<0.05); n=4.

FIG. 15. Bladder contractions induced by electrical (A and C) ormechanical (B and D) perigenital stimulation during CMGs. Electricalstimulation: 30 Hz; 15 V in A, 11-30 V in C; 0.2-ms pulse width.Mechanical perigenital stimulation (MPS): repeatedly stroking (2-3times/s) the perigenital skin with a cotton swab. The black bars underbladder pressure traces mark the stimulation duration. The calibrationbars apply to bladder pressure recordings in both A and B. Salineinfusion was started with the bladder empty. Infusion rate: 2 ml/min;n=3 for C and D.

FIG. 16. A: comparison of voided volumes induced by MPS with theresidual volumes following MPS during a 2-mo period in 3 cats. B:average bladder contraction pressure induced by MPS under isovolumetricconditions or by distention-induced micturition reflex (Reflex) is alsoshown for each cat.

FIG. 17. Comparison of average bladder contraction pressure induced byMPS or 30-Hz electrical perigenital stimulation (20-30 V) in 3 catsunder isovolumetric conditions when bladder volume was below micturitionthreshold. The thin line marks the bladder pressure of 50 cmH₂O. Thenumbers of tests are indicated in the parentheses. *Statisticalsignificance (P<0.05).

FIG. 18. Comparison of perigenital-to-bladder reflexes induced byelectrical (A and C) or mechanical (B and D) perigenital stimulationunder isovolumetric conditions. A and B: bladder volume was belowmicturition threshold. C and D: bladder volume was above micturitionthreshold and large rhythmic bladder contractions were occurring. Thecalibration bars in B apply to all bladder pressure recordings in A-D.

FIG. 19A-19D. Frequency dependent inhibition of rhythmic bladderactivity induced by electrical perianal stimulation under isovolumetricconditions. A. Effect on bladder pressure recordings at differentstimulation frequencies. The black bars under bladder pressurerecordings mark the stimulation duration. B. Area under bladder pressurecurve during stimulation. C. Average bladder contraction amplitudeduring stimulation. D. The inverse of inter-contraction interval (1/ICI)during stimulation. Bladder responses during stimulation were normalizedto the responses before stimulation in B-D. Stimulation: 30 V in A, but8-30 V in B-D; 0.2 ms pulse width. * indicates statistical significance(P<0.05). N=6 (2 tests on each cat).

FIG. 20A-20D. Intensity dependent inhibition of rhythmic bladderactivity induced by electrical perianal stimulation under isovolumetricconditions. A. Effect on bladder pressure recordings at differentintensities. The black bars under bladder pressure recordings mark thestimulation duration. B. Area under bladder pressure curve. C. Averagebladder contraction amplitude. D. The inverse of intercontractioninterval (1/ICI). Bladder responses during stimulation were normalizedto the responses before stimulation in B-D. Stimulation: 7 Hz; 0.2 mspulse width. * indicates statistical significance (P<0.05). N=6 (2 testson each cat).

FIG. 21A-21D. Bladder contraction induced by electrical perianalstimulation at different frequencies (A) or at different intensities(B). The area under bladder pressure curve is dependent on bothstimulation frequency (C) and intensity (D). Stimulation: 15 V in A, but10-30 V in C; 30 Hz in B and D; 0.2 ms pulse width. The black bars underbladder pressure traces mark the stimulation duration. The responseswere normalized to the maximal response during each trial in C-D. *indicates statistical significance (P<0.05). N=6 (2 tests on each cat).Data in A and B are from different animals.

FIG. 22A-22E. Inhibitory effect of electrical perianal stimulation onbladder activity during cystometrogram (CMG). A. Bladder pressuretraces. The initial empty bladder was infused with saline at 4 ml/min.Control: no stimulation. Stimulation: 7 Hz; 15 V; 0.2 ms pulse width.The black bar under bladder pressure trace marks the stimulationduration. B. Bladder capacity was significantly increased by electricalperianal stimulation at 7 Hz. C. The volume threshold to induce thefirst pre-micturition contraction (PMC) was also significantlyincreased. D-E. PMC amplitude and number of PMCs per minute weredecreased significantly. Responses in B-E were normalized to themeasurements during first control CMG. Stimulation in B-E: 8-30 V; 0.2ms pulse width. * indicates statistical significance (P<0.05). N=9 (3tests on each cat).

FIG. 23A-23B. Bladder contractions induced by electrical perianalstimulation during cystometrogram (CMG). Electrical stimulation: 30 Hz;25 V in A, but 11-30 V in B; 0.2 ms pulse width. The black bars underbladder pressure traces mark the stimulation duration. Saline infusionwas started with the bladder empty. Infusion rate: 4 ml/min. N=6 (2tests on each cat).

FIG. 24. Experimental setup to test the temperature influence onpudendal nerve block induced by high-frequency biphasic stimulation.EUS—External Urethral Sphincter.

FIG. 25. Pudendal nerve block by biphasic high-frequency stimulation. A.Urethral pressure induced by 10 second blocking stimulation A alone at 2mA intensity. B. Blocking stimulation A blocked urethral responsesinduced by stimulation B located centrally to the simulation A (see FIG.24); C. Blocking stimulation A failed to block urethral responses whenstimulation B was moved to a location distal to stimulation A. Blackbars in B and C indicate the stimulation durations. Temperature: 37° C.

FIG. 26. Urethral responses to high-frequency biphasic electricalstimulation of pudendal nerve at different temperatures. Stimulation: 6mA intensity, 10 sec duration. Urethral infusion rate: 2 ml/min. Theblack bars under each trace indicate the stimulation duration. Thenumber under each black bar indicates the stimulation frequency in kHz.

FIG. 27A-27B. A. Normalized urethral pressure responses change withstimulation frequency and temperature. Stimulation intensity: 1-6 mA.Urethral infusion rate: 1-2 ml/min. N=9. B. Expanded trace from FIG. 26A showing that the pressures were measured at the end of 10 secondstimulation. The black bars under the pressure trace indicate the 10second stimulation duration.

FIG. 28. A. Temperature influence on urethral response to high-frequencybiphasic electrical stimulation of pudendal nerve. Stimulation: 4 kHzfrequency, 1 mA intensity, 10 sec duration. Urethral infusion rate: 2ml/min. The black bars indicate the stimulation duration. The numberunder each black bar indicates the temperature during the time when thestimulation was applied. B. Summary of the temperature influence on thenormalized urethral pressure responses. Stimulation: 4 kHz frequency,1-6 mA intensity. Urethral infusion rate: 1-2 ml/min. N=9.

FIG. 29. Voiding induced by slow infusion of saline into the bladder ina chronic SCI cat (11 months). A: CMG without nerve stimulation. Total86 ml was infused into the bladder, but only 3 ml was voided. B: CMGwith 3-Hz stimulation of the pudendal nerve shows a suppression ofnon-voiding contractions and an increase in bladder capacity. Total 116ml was infused into the bladder, and 26 ml was voided. The black bar inB under the bladder pressure trace indicates stimulation duration.Stimulation: 3 Hz frequency, 8 V intensity, 0.2 msec pulse width.Infusion rate: 4 ml/min.

FIG. 30. Bladder capacity (A), number of non-voiding contractions (B),voiding efficiency (C), and residual volume (D) with or without 3-Hzpudendal nerve stimulation in three chronic SCI cats (N=3).Control—without 3 Hz nerve stimulation. 3 Hz—with 3—Hz nervestimulation. Stimulation: 2-10 V intensity, 0.2 msec pulse width.Infusion rate: 2-4 ml/min. * indicates statistical significance(P<0.05).

FIG. 31. Voiding induced by 20-Hz stimulation of the pudendal nerve in achronic SCI cat (9 months). Total 23 ml was infused into the bladder and18 ml was voided. The black bars under the bladder pressure traceindicate stimulation durations. Stimulation: 20-Hz frequency, 2 Vintensity, 0.2 msec pulse width. Infusion rate: 2 ml/min.

FIG. 32. Voiding induced by intermittent 20-Hz pudendal nervestimulation following 3-Hz continuous stimulation in a chronic SCI cat(9 months). Thirty-four milliliter was infused into the bladder and 32ml was voided. The black bars under the bladder pressure trace indicatestimulation durations. Stimulation: 2 V intensity, 0.2 msec pulse width.Infusion rate: 2 ml/min.

FIG. 33. Voiding efficiency (A) and residual bladder volume (B) inducedby intermittent 20-Hz pudendal nerve stimulation in three chronic SCIcats (N=3). Control—Voiding induced by bladder distension alone. 20Hz—Voiding induced by intermittent 20-Hz stimulation without prior 3-Hzcontinuous stimulation. 3 Hz+20 Hz—Voiding induced by intermittent 20-Hzstimulation with prior 3-Hz continuous stimulation. Stimulation: 2-10 Vintensity, 0.2 msec pulse width. Infusion rate: 2-4 ml/min. * indicatesstatistical significance (P<0.05).

FIG. 34. Peak bladder pressure (A) and average flow rate (B) induced bybladder distension or by intermittent 20-Hz stimulation in three chronicSCI cats (N=3). Control—Voiding induced by bladder distension. 20Hz—Voiding induced by intermittent 20-Hz stimulation. Only the peakbladder pressures and the average flow rates of the first three voidsduring a series of voids induced by intermittent 20-Hz stimulation weremeasured. Stimulation: 2-10 V intensity, 0.2 msec pulse width. Infusionrate: 2-4 ml/min. * indicates statistical significance (P<0.05).

DETAILED DESCRIPTION

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of ranges is intendedas a continuous range including every value between the minimum andmaximum values. As used herein “a” and “an” refer to one or more.

The ranges provided herein for e.g., electric pulse frequencies arebased on experimentation on cats. Nevertheless, the frequenciesnecessary to elicit a desired response in humans is very similar. Forexample, as shown in U.S. Pat. No. 7,047,078, stimulation of thepudendal nerve in human subjects at 10 Hz did not cause contractions ofthe bladder, while stimulation at 20 Hz did. As such, frequency rangesapplicable to cats are considered to be effective in humans. It shouldalso be recognized that the optimal frequency to elicit a desired effectmay vary from subject-to-subject, depending on a number of factors.Optimal frequencies to elicit the desired goals will likely need to beadjusted from person-to-person. A “subject” may be human or animal.

According to one non-limiting embodiment of the invention method ofcontrolling one or both of micturition and defecation in a subject isprovided. In one aspect the method is directed to a method of inducingmicturition and/or defecation in a subject. The method comprisesinducing one or both of micturition and defecation in the subject byapplying an electrical signal ipsilaterally or bilaterally to a pudendalnerve or a branch thereof of a subject. Either one or both (left andright) of the pudendal nerves, needs to be stimulated in this manner toproduce a contraction reflex. A typical subject has a spinal cord injuryin which voluntary control of EUS or anal sphincter, and/or otherneurological components of the micturition and/or defecation processesare diminished or absent, such as in persons with severed or damagedspinal cords or multiple sclerosis. The electrical signal compriseselectrical pulses able to create a reflex (e.g., as described above)that results in one or both of bladder contractions and rectalcontractions. Although this will cause bladder or rectal contractions,without sufficient voluntary control of the EUS and/or anal sphincter,those muscles may reflexively contract against the pressure of thecontractions, inhibiting or preventing micturition and/or defecation. Byapplying a blocking electrical signal to the pudendal nerve of thesubject or a branch thereof distal (away from the brain/spinal cord) tothe point on the nerve stimulated to produce the contraction reflexes,and preferably to both (left and right) pudendal nerves to completelyblock reflexive contraction of the EUS and/or anal sphincter. Theblocking electrical signal is able to inhibit contraction of one or bothof the external urethral sphincter and the anal sphincter of thesubject. As will be recognized by a person of skill in the art,characteristics of electrical pulse, including, without limitation,amplitude (pulse strength, referring to the magnitude or size of asignal voltage or current), voltage, amperage, duration, frequency,polarity, phase, relative timing and symmetry of positive and negativepulses in biphasic stimulation, and/or wave shape (e.g., square, sine,triangle, sawtooth, or variations or combinations thereof) may be variedin order to optimize results in any particular subject or class ofsubjects. Subjects may be classified by species, disease/condition, sex,or any other factor that can be generalized to a group.

The method may further comprise, prior to (and/or after) producing oneor both of micturition and defecation in the subject, applying anelectrical signal to a pudendal nerve or a branch thereof of thesubject, the electrical signal having an amplitude and frequency able tocreate a reflex that inhibits one or both of bladder contractions andrectal contractions in the subject. This produces a storage stage,similar to the typical storage stage of the normal micturition ordefecation processes.

The primary characteristic of the electrical signals used to produce adesired response, as described above, is pulse frequency. Althougheffective ranges (e.g., frequencies able to produce a stated effect) mayvary from subject-to-subject, and the controlling factor is achieving adesired outcome, certain, non-limiting exemplary ranges may be asfollows. For stimulating bladder, rectal or sexual gland/musclecontractions, those frequencies may range from approximately 15 Hz(Hertz, or pulses per second) to approximately 50 Hz. For blocking nervefunction, such as in blocking reflexive contraction of the EUS or analsphincter, those frequencies range from approximately 4 kHz (kiloHertz,e.g., 4,000 Hz) or greater, for example and without limitation, fromapproximately 4 kHz to approximately 10 kHz. For inhibiting contractionof the bladder or rectum, those frequencies may range from approximately0.5 Hz to approximately 15 Hz. Variations from these ranges may becapable of achieving the same results. Stated ranges are intended toinclude all values and ranges within the stated ranges. So long as othercharacteristics of the electrical signals (e.g., without limitation,amplitude, voltage, amperage, duration, polarity, phase, relative timingand symmetry of positive and negative pulses in biphasic stimulation,and/or wave shape) are within useful ranges, modulation of the pulsefrequency will achieve a desired result. Useful values for those othercharacteristics are well-known in art and/or can be readily establishedby routine experimentation, for instance by the ability to prevent orinduce voiding, for instance, by methods described herein.

Wedensky inhibition, anodal block, depolarizing prepulses and slowlyrising pulses are non-limiting examples of art-recognized methods usefulin blocking nerves. So long as other pulse parameters are withinacceptable limits, the blockage is temporary and does not damage theblocked nerve. In one non-limiting embodiment, nerve blockage isachieved by applying middle-range currents of at least 4 kHz, forexample and without limitation, in the range of from 4 kHz to 10 kHz tothe nerve using a suitable electrode. The maximum useful frequency is afrequency that is able to achieve the desired result (e.g., nerveblockage) with acceptable safety. The voltage and amperage of thecurrent typically should be below 15 mA and 50V to prevent nerve damage,though typically lower values are applied, for example in the range of 1to 30V.

In another embodiment, a system for controlling one or both ofmicturition and defecation in a subject is provided. The systemcomprises an implantable pulse generator unit having a first outputchannel adapted to produce electric pulses in a first stage at afrequency ranging from 0.5 Hz to 15 Hz, and in a second stage at afrequency ranging from 15 Hz to 50 Hz, and a second output channeladapted to produce electric pulses at a frequency of greater thanapproximately 4 kHz, for example and without limitation, between 4 kHzand 10 kHz during the second stage, for nerve blockage, for exampleprevention of reflexive contractions of the EUS and/or anal sphincterwhen applied to the pudendal nerve. The pulse generation unit maycomprise a first wireless communication system for receiving controlinstructions from a wireless controller; and a wireless controller,comprising an input, a display and a second wireless communicationsystem configured to send control instructions to the implantable pulsegenerator. In one embodiment, the electric pulses are biphasic. Thefirst wireless communication system may also transmit status informationfor the pulse generator to the wireless controller. The pulse generatorunit may also further comprise a third output channel adapted to produceelectric pulses at a frequency of at least 4 kHz, for example andwithout limitation in a range between 4 kHz and 10 kHz, at the same timeas the second output channel. Further description of one embodiment ofsuch a system is described in reference to FIG. 1. The phrases“configured to” and “adapted to” and like terms or phrases refer to themanufacture, production, modification, etc. of a device or system toproduce a desired function. In the context of the devices or systemsdescribed herein, a device or system “adapted to” or “configured to”produce a desired output is a device programmed of otherwisemanufactured, produced, modified, etc. in any manner to produce thestated effect.

In another embodiment, voiding, defecation, ejaculation, orgasm,inhibition of bladder or rectal contractions, reduction of pelvic painand/or sexual stimulation may be accomplished by stimulating asuperficial (close to the skin) branch of a pudendal nerve. As describedabove, many of these results can be accomplished by stimulating thepudendal nerve or a branch thereof by implanted electrodes. However,previously it was not known if this could be accomplished by superficialstimulation of branches of the pudendal nerve. By superficialstimulation, it is meant by applying electrical stimulus to the skin,intradermally or subdermally. The benefit of this is that surgicalimplantation of electrodes, and the requisite surgical skill and expenseis not necessary. Block of the pudendal nerves is not possible by thismethod, but effective voiding may be achieved, for instance, as depictedbelow, by stimulating the nerves in a pulsatile fashion. The electricalpulses will stimulate both smooth muscle contractions in the bladder,rectum and glands and muscles associated with ejaculation, orgasm orsexual stimulation, as well as the EUS and anal sphincter. Because thestriated muscle of the EUS and anal sphincters will relax upon cessationof electrical stimulation before the smooth muscle of the bladder,rectum and sexual glands and ducts, voiding of the bladder, rectum andsexual glands and ducts is achieved for a short time period followingcessation of the electrical pulse (post-stimulus voiding).

In this embodiment of the method, electrodes are placed perigenitally orperianally in order to be in sufficient proximity to the pudendalnerves. Electrodes may be placed on the skin, intradermally orsubdermally, by any useful method, prior to stimulus. In one embodiment,trans-epidermal nerve stimulation (TENS) may be applied, and therefore,and electrode, re-usable, disposable, or semi-disposable, that is usefulin TENS applications may be suitable for applying stimulus tosuperficial branches of the pudendal nerve as described herein. Pulseamplitude, voltage, amperage, duration, waveform, phase, etc. may bevaried to achieve optimal activity. For example, pulses ranging fromapproximately 15 Hz to 50 Hz may be applied for 0.1 to 60 seconds, orranges therebetween, including 1, 2, 5, 10, 15, 20, 25, 30, 45 or 50seconds. Bladder or rectal voiding will occur immediately followingcessation of the stimulus. When voiding ceases, in for example, 1 to 20seconds, pulses may again applied until post-stimulus voiding ceases. Itmay be desirable to minimize pulse lengths. For inhibitory and painrelief effects, for example and without limitation, pulses ranging from0.5 Hz to 15 Hz may be applied.

Damage to nerves by the application of an electrical current may beminimized, as is known in the art, by application of biphasic pulses orbiphasic waveforms to the nerve(s), as opposed to a monophasic pulses orwaveforms that can damage nerves in some instances of long-term use.“Biphasic current,” “biphasic pulses” or “biphasic waveforms” refer totwo or more pulses that are of opposite polarity that typically are ofequal or substantially equal net charge (hence, biphasic and chargebalanced). This is accomplished, for example, by applying through anelectrode one or more positive pulses, followed by one or more negativepulses, typically of the same amplitude and duration as the positivepulses, or vice versa, such that the net charge applied to the target ofthe electrode is zero or approximately zero. The opposite polaritypulses may have different amplitudes, profiles or durations, so long asthe net applied charge by the biphasic pulse pair (the combination ofthe positive and negative pulses) is approximately zero.

The electrodes used to stimulate the nerves may be of any usefulcomposition or mixture of compositions, such as platinum or stainlesssteel, as are known in the art, and may be of any useful configurationfor stimulating nerves, including monopolar, bipolar, or tripolarelectrode with or without a cuff wrapping around the pudendal nerve, asare known in the art.

Pulses applied to nerves via electrodes are generated by any devicecapable of delivering suitable electrical pulses. For uses, such asperianal or perigenital stimulation of pudendal nerve branches, thedevice can have one or more output channels. In the context of devicesand methods used herein, an output channel is an electrical circuit thatis able to send an electrical signal to an electrode. If a device hastwo or more output channels, the output of each channel is typicallyindependently controllable with regard to one or more parameters, suchas pulse frequency, amplitude, voltage, amperage, duration, polarity,phase, relative timing and symmetry of positive and negative pulses inbiphasic stimulation, and/or wave shape. In the context of theembodiments for blocking the pudendal nerves or branches thereof, whilestimulating contractions of the bladder or rectum, a device having twoor three independent output channels is needed. A first channel is usedto provide an electrical signal suitable for inducing bladder and/orrectum contractions during a voiding stage, such as, without limitation,biphasic pulses of between 15 Hz and 50 Hz, and an electrical signalsuitable for inhibiting bladder and/or rectum contractions during astorage stage, such as, without limitation, biphasic pulses of between0.5 Hz and 15 Hz. A second, or a second and third output channel may beused to provide an electrical signal suitable for blocking activity ofthe EUS and/or anal sphincter during the voiding stage, such as, withoutlimitation, biphasic pulses of at least 4 kHz, for example and withoutlimitation, between 4 kHz and 10 kHz. The output of the second outputchannel may be split to send signals to both sides of the pudendalnerves or branches thereof, or the output of a second output channel anda third output channel may be independently sent to left and rightpudendal nerves or branches thereof.

FIG. 1 depicts schematically one non-limiting embodiment of athree-channel system for stimulating pudendal nerves or branches thereofaccording to the methods described herein. Pulse generator 10 isdepicted as having three output channels. Wire leads 12 and 13 areattached to electrodes 14 and 15, which are placed about pudendal nerve20. Electrode 15 is shown distal to electrode 14. In the context of amethod comprising blocking the EUS and/or anal sphincter and stimulatingor inhibiting contractions in the bladder and/or rectum, electrode 14would be used to stimulate or inhibit bladder and/or rectalcontractions, while electrode 15 would be used to block the EUS or analsphincter. As needed, wire lead 16 is attached to another electrode (notshown in FIG. 1) that is placed about the pudendal nerve on the otherside of the body to block the pudendal nerve on the opposite side. Pulsegenerator 10 is shown as implanted beneath skin 40. Output parameters ofthe pulse generator 10 can be controlled via a wired interface, butpreferably is controlled by wireless transmission, which can be carriedany suitable wireless protocol, such as radio frequency, IEEE802.11a/b/g, Bluetooth, etc. Thus, an external controller 30 is depictedfor communicating with the pulse generator 10. External controller 30 isdepicted as having a display 32, such as an LCD, LED or OLED display,and a keypad 34 for entering data into the external controller 30.External controller is depicted as sending a wireless transmission 36 topulse generator 10, though in another embodiment, data can betransferred both to the pulse generator 10 from the externalcommunicator 30 and vice-versa, to permit monitoring of one or moreparameters of pulse generator 10, including, without limitation, outputsignal characteristics (e.g., frequency, amplitude, etc. as outlinedabove) and battery strength. Activity of pulse generator 10 and externalcontroller 30 typically is microprocessor controlled andsoftware/firmware installed onto the pulse generator 10 and externalcontroller 30 hardware may be used to implement the described tasks, andto provide, for example and without limitation, a GUI (graphical userinterface) for the display 32, which facilitates use of the system. Bothpulse generator 10 and external controller 30 may comprise any suitableelectrical and electronic components to implement the activities,including, microprocessors, memory (e.g., RAM, ROM. Flash memory, etc.),connectors, batteries, power transformers, amplifiers, etc. A person ofskill in the electronic arts will be able to implement such a systemusing readily-available electronics parts and ordinary programmingskills. Proprietary chips, chipsets, etc. may be designed andmanufactures to implement the devices described herein.

External controller 30 may be a proprietary device that is specificallydesigned for the task, or a non-proprietary device, such as a smartphone or a portable computer. Pulse generator 10 may comprise any numberof channels, so long as the number of channels needed to implement adesired method is provided. In one variation of the embodiment depictedin FIG. 1, wire lead 13 is split into two wire leads, each of which areterminated in a separate electrode, with one wire lead and electrode forstimulating a left pudendal nerve or branch thereof, and the other wirelead for stimulating a right pudendal nerve or branch thereof.

One potential difficulty with use of wireless devices is one ofidentity. A controller should only be able to control one pulsegenerator to prevent accidental stimulation of unintended subjects, oreven intentional stimulation. In its simplest form, the transmissionrange of the devices can also be limited to prevent transmission overdistances more than a few feet, thereby limiting the chances ofunintended stimulation (crosstalk). Also, any number of identityverification mechanisms may be utilized to prevent crosstalk. In oneembodiment, different transmission wavelengths may be used for differentdevices, thus lowering the likelihood of crosstalk. In anotherembodiment, the pulse generator is programmed to only respond to atransmission containing a pre-defined signal, such that the pulsegenerator and external wireless controller must first, and/orperiodically “handshake” in order to communicate. Likewise, the pulsegenerator and/or controller may transmit encrypted signals which onlycan be decrypted by a key stored in the other of the pulse generatorand/or controller. In another embodiment, RFID tagging technology may beused to ensure that the controller and pulse generator match. Anycombination of these proximity and/or identity verification measures maybe used to prevent cross-talk. Other useful technologies for ensuringsecurity and identity in communication are, or may be available and areequally applicable.

Also provided herein is a method of stimulating a physiological responsein a subject. The method comprises stimulating the pudendal nerve or abranch thereof in a subject using an implanted electrode with electricalpulses at a frequency and amplitude able to either inhibit or stimulateone or more of bladder contractions, rectum contractions, andbulbospongiosus and ischiocavernosus muscle contractions, therebyobtaining the physiological response. The physiological response may beone or more of micturition, defecation, ejaculation, orgasm, inhibitionof bladder contractions, inhibition of pelvic pain of bladder, urethra,prostate, anus, or rectum, inhibition of rectal contractions, and sexualarousal. In one embodiment, the electrical pulses range from 0.5 to 15Hz, which is suitable for is inhibition of bladder contractions, andinhibition of pelvic pain of bladder, urethra, prostate, anus, orrectum, and inhibition of rectal contractions, including, withoutlimitation pain from interstitial cystitis/painful bladder syndrome,which is treatable by electrical stimulation. In another embodiment, theelectrical pulses range from 15 to 50 Hz, in which case thephysiological response is, without limitation, micturition, defecation,ejaculation, and one or both of orgasm and increased sexual response.The 15-50 Hz pulses may be applied intermittently, for example andwithout limitation, in two or more stimulation intervals of from 0.5 to60 seconds with a rest period of no electrical stimulation able to causebladder or rectal contractions between stimulation intervals. Typicallyduring the rest period, no inhibitory stimulus (e.g., stimulus in therange of 0.5 to 15 Hz) is applied. During the rest period no electricalsignal, or essentially no electrical signal is applied.

Also provided is a system for controlling one or both of micturition anddefecation in a subject. The system comprises a pulse generator unithaving a first output channel adapted to produce electric pulses in afirst stage at a frequency ranging from 0.5 Hz to 15 Hz, and in a secondstage at a frequency ranging from 15 Hz to 50 Hz and a controllerconfigured to send control instructions to the implantable pulsegenerator. The pulses may be biphasic and, typically, balanced. Thesystem, especially when in use, comprises a lead wire and an electrodeconnected to the output channel, with the electrode placed either on asubject's skin to stimulate a superficial branch of a pudendal nerve,such as perianally or perigenitally, or implanted, in which case theelectrode stimulates the pudendal nerve or a branch thereof. In anyembodiment in which a pulse generator is implanted, the device is“implantable, which means that it is medically acceptable forimplantation. A wireless controller, as in any embodiment describedherein, may be used to control an implanted device. For externalelectrodes, the pulse generator and controller may be housed separatelyor within a single housing. Variations of device/system structure forany device or system described herein will be apparent to one of skillin the art and are a matter of design choice and optimization within theabilities of a person of ordinary skill in the art.

Example 1

The following determined if an efficient voiding reflex can be inducedin the chronic SCI cats by a continuous 20 Hz pudendal nervestimulation. We used a nerve blocking technique to determine if detrusorsphincter dyssynergia interferes with pudendal nerve stimulation-inducedvoiding. Our previous studies (Tai C, Roppolo J R, de Groat W C. Blockof external urethral sphincter contraction by high frequency electricalstimulation of pudendal nerve. J Urol 2004; 172:2069-72 and Tai C,Roppolo J R, de Groat W C. Response of external urethral sphincter tohigh frequency biphasic electrical stimulation of pudendal nerve. J Urol2005; 174:782-6) revealed that electrical stimulation of the pudendalnerve in cats at a frequency of 6-10 kHz blocks nerve conduction. Inthis study, combining excitatory and blocking stimulation of thepudendal nerves induced an efficient voiding reflex in chronic SCI cats.

Materials and Methods

A total of six female cats were used in this study (three spinal intactand three chronic SCI animals, 3.5-4.5 kg). For the three chronic SCIcats that were also used in our previous study, 35 spinal cordtransection was performed (3-11 months prior to the experiment) at theT9-T10 spinal cord level by a dorsal laminectomy under isofluraneanesthesia and aseptic conditions. During the experiments, animals (bothspinal intact and SCI) were anesthetized with α-chloralose (60 mg/kgi.v., supplemented as needed) following induction with halothane (2-3%in O₂). Systemic blood pressure was monitored via a cannula placed inthe right carotid artery. A tracheotomy was performed and a tube wasinserted to secure the airway. A catheter for i.v. infusion wasintroduced into right ulnar vein. A double lumen catheter (five French)was inserted into the bladder via the dome and secured by a ligature(see FIG. 2A). One lumen of the catheter was attached to a pump toinfuse the bladder with saline, and the other lumen was connected to apressure transducer to monitor the bladder activity. A funnel was usedto collect the voided fluid in a beaker that was attached to a forcetransducer to record the volume. For the three chronic SCI animals, thepudendal nerves were accessed posteriorly between the sciatic notch andthe tail. Two tripolar cuff electrodes [Micro Probe, Inc., Gaithersburg,Md.; NC223(Pt)] were placed around the left pudendal nerve (Elec. #1 and#2 in FIG. 2A). A third tripolar cuff electrode was placed around theright pudendal nerve (Elec. #3 in FIG. 2A). The three electrode leads ineach cuff electrode were made of platinum wires (diameter 0.25 mm) witha 2 mm distance between the leads. The two leads at each end of the cuffelectrode were connected together (see FIG. 2A). After implanting thepudendal nerve electrodes, the muscle and skin were closed by sutures.For the three spinal intact animals, electrodes were not implanted onthe pudendal nerve. The temperature of the animals was maintained at35-37° C. during the experiments using a heating pad. A pulse oximeter(Nonin Medical, Inc., Plymouth, Minn.; 9847V) with its sensor clipped onthe tongue of animals was used to monitor the arterial oxygen saturationand heart rate. Blood pressure, heart rate, and front paw withdrawreflex were used to evaluate the anesthetic depth.

In the experiments in chronic SCI cats, uniphasic pulses at 20 Hzfrequency, 2-10 V intensity and 0.2 msec pulse width were used tostimulate the pudendal nerve at electrode #1 (see FIG. 2B) in order toinduce the excitatory pudendal-to-bladder reflex and bladdercontractions. Our previous study 34 in chronic SCI cats showed that 20Hz pudendal nerve stimulation induces strong bladder contractions. Thestimulation intensity was determined at the beginning of each experimentby a preliminary test of its effectiveness to induce bladdercontractions. In order to block pudendal nerve conduction and prevent anEUS contraction, a train of high-frequency, biphasic, continuous (dutycycle 100%), charge-balanced, rectangular pulses at 10 kHz frequency and10 mA intensity were delivered to electrode #2 and/or #3 (see FIG. 2B).The stimulation frequency and intensity were shown in our previousstudies (Id.) to be effective in blocking the pudendal nerves of cats. AGrass S88 stimulator (Grass Medical Instruments) with stimulus isolator(Grass Medical Instruments, SIU5) was used to generate the uniphasicstimulus pulses for electrode #1. The high-frequency, biphasicstimulation waveforms (10 kHz) used at electrodes #2 and #3 weregenerated by a computer with a digital-to-analog circuit board (NationalInstruments, Austin, Tex.; AT-AO-10) that was programmed using LabViewprogramming language (National Instruments). Linear stimulus isolators(World Precision Instruments, Sarasota, Fla.; A395) were used to deliverthe high-frequency, biphasic constant current pulses to the nerves viaelectrodes #2 and #3.

Starting with the bladder empty, saline was slowly infused (0.5-4ml/min) into the bladder to induce a voiding reflex (i.e., acystometrogram—CMG). Bladder capacity was defined as the infused volumeat which a bladder contraction was induced and fluid was released fromthe urethral orifice. When fluid was released, the infusion was stopped.The distension induced voiding was evaluated in both spinal intact andchronic SCI cats. In the chronic SCI cats during an intercontractionquiet period after stopping the bladder infusion, 20 Hz stimulation wasapplied to the pudendal nerve to induce bladder contractions. In anotherstimulation paradigm, prior to the start of 20 Hz stimulation atelectrode #1, the 10 kHz blocking stimulation was applied either toelectrode #2 only or to electrodes #2 and #3 (see FIG. 2B) in order toblock the EUS contraction during the 20 Hz pudendal nerve stimulation.Voiding efficiency, maximal bladder pressure, and average flow rate weremeasured in order to evaluate the effectiveness of the induced voidingreflex. Voiding efficiency is defined as the total voided volume dividedby the total infused volume. Parameters measured from multiple trials inthe same animal were averaged and then presented as mean±standard error(SE). Since ANOVA analysis showed no significant difference betweentrials in the same animals, two-way ANOVA (experimental conditions vs.animals) was used to determine any statistical significance (P<0.05).Since each parameter was measured multiple times in the same animal, foreach experimental condition there were three data sets (mean, SD, and N)from the three animals Two-way ANOVA was performed on the three datasets (animals) for different experimental conditions (control, nervestimulation and block, etc.).

Results

Voiding Reflex in Normal and Chronic SCI Cats As shown in FIG. 3, theCMGs in spinal intact and chronic SCI cats were markedly different.During bladder filling in chronic SCI cats the bladder exhibitedmultiple, low amplitude, short duration, non-voiding contractions (i.e.,neurogenic detrusor overactivity, FIG. 3B); whereas the bladder ofspinal cord intact animals was quiescent until the onset of voiding(FIG. 3A). Voiding was also different.

Compared to the bladder contractions induced by bladder distension inspinal cord intact cats the contractions induced by distension inchronic SCI cats were weaker and considerably less efficient inproducing voiding (FIGS. 3A and 3B). On average the peak intravesicalpressures during reflex contractions in chronic SCI cats were only 30%of those in spinal intact cats (FIG. 4A, 23.1±1.7 cmH2O vs. 72.5±11.8cmH₂O, P<0.05). The average duration of contractions was only 20% of theduration in spinal intact animals (21.9±0.9 sec vs. 109.3±8.2 sec,P<0.05). Average voiding efficiency (7.3±0.9%, FIG. 5) and flow rate(0.23_0.07 ml/sec, FIG. 4B) were markedly (P<0.05) lower than the valuesin spinal intact cats (93.6±2.0% and 0.56±0.16 ml/sec respectively, seeFIGS. 4B and 5). Note that in both types of animals the saline infusionwas stopped when fluid was released from the urethral meatus.

To determine if voiding might improve as bladder volume increased withcontinued infusion in chronic SCI cats the CMGs were also continuedbeyond the time of the first void (FIG. 6A). This only produced a seriesof short duration, small bladder contractions of approximately the sameamplitude and duration. Each contraction released only a small amount offluid equivalent to the volume infused during each contraction interval.During the experiment shown in FIG. 6A the residual bladder volumeremained static during the series of small voiding reflexes. At the endof 12 min infusion beyond the first void, the residual bladder volumewas almost equal to the volume (19 vs. 20 ml) prior to the first void.Thus in chronic SCI cats continued infusion of fluid did not increasebladder volume; and voiding efficiency during each void was still poor.

A second approach to improve voiding efficiency was to increase bladdervolume prior to voiding utilizing a low frequency pudendal nervestimulation (3 Hz) which we showed in a previous study (Tai C, Smerin SE, de Groat W C, et al. Pudendal-to-bladder reflex in chronicspinal-cord-injured cats. Exp Neurol 2006a; 197:225-34) suppressednon-voiding contractions during bladder filling and increased bladdercapacity in chronic SCI cats. It was anticipated that at a largerbladder volume a more prolonged and larger amplitude bladder contractionmight occur. However as shown in FIG. 6B in the same chronic SCI catused in FIG. 3B, 3 Hz pudendal nerve stimulation inhibited bladderactivity during filling and increased bladder capacity from 74 to 124ml, but after the stimulation was stopped only short duration bladdercontractions occurred and voiding was still inefficient (31 ml voidedleaving 93 ml residual volume in the bladder).

Voiding Reflex in Chronic SCI Cats Induced by Pudendal Nerve Stimulationand Block

As shown in FIG. 7, 20 Hz excitatory pudendal nerve stimulation appliedto electrode #1 (see FIG. 2) elicited large amplitude (40 cmH₂O), longduration (80-100 sec) bladder contractions in chronic SCI cats that wereequivalent to the voiding contractions in cats with an intact spinalcord. However, voiding did not occur during the 20 Hz stimulation. Onthe other hand, when 10 kHz blocking stimulation was applied for a briefperiod (20 sec) to the pudendal nerves bilaterally (electrodes #2 and#3, see FIG. 2) during the 20 Hz excitatory stimulation, the bladderpressure immediately decreased and voiding occurred (FIG. 7). When theblocking stimulation was stopped, voiding stopped and bladder pressureincreased. The bladder pressure was maintained until the end of 20 Hzstimulation when an additional void occurred presumably due to the rapidrelaxation of the urethral sphincter while the bladder pressure wasstill high. An efficient voiding reflex was also induced in chronic SCIcats by the 20 Hz pudendal nerve stimulation when the 10 kHz blockingstimulation was applied ipsilaterally (i.e., only applied to theelectrode #2, see FIG. 2) or bilaterally (i.e., applied to bothelectrodes #2 and #3, see FIG. 2) prior to the excitatory pudendal nervestimulation. FIG. 8A shows in a chronic SCI cat that a large amount (53ml) of fluid was voided from a bladder containing 86 ml when the 20 Hzstimulation was combined with the ipsilateral 10 kHz blockingstimulation resulting in a voiding efficiency of 61.6%. The voidingoccurred primarily during the first stimulation period when two periodsof stimulation were used. FIG. 8B shows in another chronic SCI cat thatthe 20 Hz stimulation combined with bilateral 10 kHz blockingstimulation also induced an efficient (88%) voiding reflex. As shown inFIG. 8B, a small amplitude bladder contraction was induced at the onsetof the 10 kHz blocking stimulation without voiding. When the 20 Hzstimulation was started, the bladder pressure increased quickly and thendecreased once fluid started to flow through the open urethra. Duringthe induced voiding the fluid flow was a steady stream and the bladderpressure was relatively low (less than 30 cmH₂O).

On average the 20 Hz pudendal nerve stimulation coupled with 10 kHzipsilateral block significantly (P<0.05) increased the voidingefficiency in chronic SCI cats to 73.2±10.7% compared to the voidingefficiency induced by bladder distension (7.3±0.9%) as shown in FIG. 5.With 10 kHz bilateral block of the pudendal nerves, the 20 Hz pudendalnerve stimulation further increased voiding efficiency to 82.5±4.8%.This efficiency is not significantly different from the voidingefficiency in spinal intact cats induced by bladder distension, althoughthe voiding efficiency induced by ipsilateral block is stillsignificantly different (FIG. 5). As shown in FIG. 4A, the maximalbladder pressure (31.4±6.4 cmH₂O) during voiding induced by the 20 Hzpudendal nerve stimulation with 10 kHz bilateral nerve block wassignificantly (P<0.05) increased in chronic SCI cats compared to thebladder distension induced voiding (23.1±1.7 cmH₂O). But it was stillsignificantly (P<0.05) lower than that in spinal intact cats (72.5±11.8cmH₂O). Therefore, simultaneously stimulating and blocking the pudendalnerves induced low pressure voiding in chronic SCI cats. The averageflow rate (0.68±0.08 ml/sec) was significantly (P<0.05) increased by the20 Hz pudendal nerve stimulation with bilateral 10 kHz block in chronicSCI cats compared to the distension induced voiding (0.23±0.07 ml/sec)(see FIG. 4B). It was not significantly different (P>0.05) from the flowrate in spinal intact cats (0.56±0.16 ml/sec). Therefore, in chronic SCIcats 20 Hz pudendal nerve stimulation combined with 10 kHz nerve blockinduced a markedly different voiding reflex than bladder distension (seeFIGS. 3B, 6, and 8) resulting in an efficient voiding (FIG. 5) with afast flow rate (FIG. 4B) and a low bladder pressure (FIG. 4A).

Discussion

This study revealed marked differences in voiding reflexes inchloralose-anesthetized cats with an intact spinal cord and in chronicSCI cats (FIGS. 3-6). Chronic SCI cats exhibited a reduced voidingefficiency and urethral flow rate that is attributable to low amplitudeand short duration reflex bladder contractions as well as to poorcoordination between the urinary bladder and the EUS. Electricalstimulation of afferent axons in the pudendal nerve reversed the defectin bladder contractions, eliciting large amplitude, long duration,reflex bladder contractions but still did not improve voiding efficiencydue to simultaneous activation of motor pathways to the EUS (FIG. 7).However when the motor pathways were blocked by high-frequencystimulation (10 kHz) of the pudendal nerves unilaterally or bilaterally,voiding efficiency markedly improved (FIGS. 5 and 8). These resultsraise the possibility that combined pudendal afferent nerve stimulationand efferent nerve block might be useful in promoting voiding in peoplewith SCI.

Voiding Reflex in Normal Cats Versus Voiding Reflex in Chronic SCI Cats

In normal cats, the pontine micturition center (PMC) located in therostral pons coordinates bladder and EUS activity during voiding. Duringbladder filling when afferent input to the PMC reaches the threshold fortriggering micturition (i.e., bladder capacity) descending projectionsfrom the PMC induce a sustained bladder contraction with a simultaneousEUS relaxation resulting in release of a large volume of fluid from thebladder (see FIG. 3A). This spinobulbospinal voiding reflex produces afast flow rate (FIG. 4B) with a high voiding efficiency (FIG. 5).However, in chronic SCI cats the contribution of the PMC is lost andvoiding is mediated purely by a spinal reflex. This spinal voidingreflex can only induce a series of small, short-lasting bladdercontractions (FIGS. 3B, 4A, and 6) when the bladder volume reaches athreshold level, but only a very small percentage of bladder volume(FIG. 5) is released at a slow flow rate (FIG. 4B). The inefficiency ofvoiding in chronic SCI cats might be attributable to two differentspinal reflexes. One is the bladder-to-bladder spinal reflex, and theother is the bladder-to-pudendal spinal reflex. The bladder-to-bladderspinal reflex is brief (see FIGS. 3B and 6) and generates smallerbladder pressures (see FIG. 4A) compared to the spinobulbospinal reflexin spinal intact cats. In awake, chronic SCI cats, similar shortduration, small amplitude bladder contractions were also observed duringvoiding. This indicates that the bladder-to-bladder spinal reflex isvery different from the bladder-to-bladder supraspinal reflex mediatedby the PMC.

Once it is triggered, the supraspinal reflex can maintain a largeamplitude bladder contraction and sustain the bladder pressure as thebladder volume becomes smaller during the voiding (FIG. 3A). Thissustained bladder contraction is driven by a constant input from the PMCto the parasympathetic neurons in the sacral spinal cord. However, thebladder-to-bladder spinal reflex lacks of this sustained input. Instead,the bladder-to-bladder spinal reflex is directly driven by the tensionreceptors in the bladder wall. Once voiding occurs, and bladder volumeand tension in the bladder wall are reduced, attenuation of afferentinput to spinal cord seems to turn off the reflex (FIGS. 3B and 6).Therefore, the bladder-to-bladder spinal reflex cannot sustain thebladder contraction as the bladder volume declines during voiding. Inaddition to the weak bladder-to-bladder spinal reflex, voidinginefficiency in chronic SCI cats is also caused by a bladder-to-pudendalspinal reflex. This reflex triggers EUS contractions and increasesurethral outlet resistance during a bladder contraction (i.e., detrusorsphincter dyssynergia), which further reduces the ability of thedistension-induced, small, transient bladder contractions to eliminatefluid from the bladder (see FIGS. 3B and 6).

Although it seems unlikely that the surgical manipulation on thepudendal nerves caused the small, transient bladder contractions in thechronic SCI cats, it is worth noting that the pudendal nerves in normalanimals were not surgically manipulated and this might have contributedto the differences in bladder activity between normal and SCI cats. Thusthe factors contributing to the differences between thebladder-to-bladder spinal reflex in chronic SCI cats and thebladder-to-bladder spinobulbospinal reflex in normal cats need to befurther investigated.

Pudendal-to-Bladder Spinal Reflex

In this study, we have demonstrated that an excitatorypudendal-to-bladder spinal reflex exists in chronic SCI cats. Thisspinal reflex can generate sustained bladder contractions (FIG. 7)strong enough to induce efficient voiding if the EUS contraction can beprevented (FIG. 5 and FIG. 8). The 20 Hz pudendal nerve stimulationprovides a sustained excitatory input to the sacral parasympatheticneurons that is similar to what is provided by the PMC. However, itappears that this excitatory pudendal-to-bladder spinal reflex is alsodependent on bladder volume. At a smaller bladder volume, it becomesweaker (see FIG. 7). Thus it is likely that there is a positiveinteraction between pudendal and bladder afferent inputs to the spinalmicturition reflex circuitry. The excitatory pudendal-to-bladder spinalreflex could be induced during distal blockade of the pudendal nerve(see FIG. 8) indicating that this excitatory spinal reflex is activatedby stimulating the afferent fibers in the pudendal nerve rather than theefferent fibers.

The pudendal-to-bladder spinal reflex in chronic SCI cats can be eitherexcitatory or inhibitory depending on the stimulation frequency.Previous studies (Walter J S, Wheeler J S, Robinson C J, et al.Inhibiting the hyperreflexic bladder with electrical stimulation in aspinal animal model. Neurourol Urodyn 1993; 12:241-53; Mazieres L, JiangC, Lindstrom S. Bladder parasympathetic response to electricalstimulation of urethral afferents in the cat. Neurourol Urodyn 1997;16:471-2; Sundin T, Carlsson C A, Kock N G. Detrusor inhibition inducedfrom mechanical stimulation of the anal region and from electricalstimulation of pudendal nerve afferents: An experimental study in cats.Investigative Urol 1974; 11:374-8; Fall M, Erlandson B E, Carlsson C A,et al. The effect of intravaginal electrical stimulation on the felineurethra and urinary bladder: Neuronal mechanisms. Scand J Urol Nephrol1978; 44:19-30; and Lindstrom S, Fall M, Carlsson C A, et al. Theneurophysiological basis of bladder inhibition in response tointravaginal electrical stimulation. J Urol 1983; 129:405-10) showed inboth normal and SCI cats that an inhibitory pudendal-to-bladder reflexwas induced by pudendal nerve stimulation at a frequency below 10 Hz(see also FIG. 6B). However, as demonstrated in this and our previousstudy (Tai C, Smerin S E, de Groat W C, et al. Pudendal-to-bladderreflex in chronic spinal-cord-injured cats. Exp Neurol 2006a;197:225-34), the pudendal-to-bladder spinal reflex in chronic SCI catsbecomes excitatory at a stimulation frequency of 20 Hz. Although themechanism of this frequency dependence is unknown, it is clear thatdifferent stimulation frequencies must activate different spinalmicturition reflex circuitry. Barrington (Barrington F J F. Thecomponent reflexes of micturition in the cat, Parts I and II. Brain1931; 54:177-88 and Barrington F J F. The component reflexes ofmicturition in the cat, Parts III. Brain 1941; 64:239-43) identified aspinal reflex from urethra to bladder when urine flows through theurethra. He showed that this reflex can induce a bladder contractionwhen bladder volume is high. Meanwhile, Garry et al. (Garry R C, RobertsT D, Todd J K. Reflexes involving the external urethral sphincter in thecat. J. Physiol 1959; 149:653-65) reported that fluid flowing throughthe urethra could inhibit bladder activity when bladder volume was low.Since the urethra is innervated by the pudendal nerve, pudendal nervestimulation at different stimulation frequencies might trigger thedifferent urethra-to-bladder reflexes.

In acute SCI cats (i.e., within hours after spinal cord transection),pudendal nerve stimulation induced small (less than 20 cmH₂O) bladdercontractions. (Boggs J W, Wenzel B J, Gustafson K J, et al. Spinalmicturition reflex mediated by afferents in the deep perineal nerve. JNeurophysiol 2005; 93:2688-97 and Shefchyk S J, Buss R R. Urethralpudendal afferent-evoked bladder and sphincter reflexes in decerebrateand acute spinal cats. Neurosci Lett 1998; 244:137-40) However, thepudendal-to-bladder spinal reflex in acute SCI cats is excitatoryregardless of the frequency of pudendal nerve stimulation, (Boggs J W,et al., J Neurophysiol 2005; 93:2688-97) which is very different fromthe effects in chronic SCI cats. This difference between acute andchronic SCI cats may be due to the fact that neuroplasticity in thespinal cord which underlines the emergence of excitatorybladder-to-bladder and pudendal-to-bladder spinal reflexes requiresseveral weeks to fully develop. Neurogenic detrusor overactivity anddetrusor sphincter dyssynergia that are indicators of spinalreorganization after SCI exist in chronic SCI animals and humans, but donot exist after acute SCI. Instead, acute SCI results in detrusorareflexia (i.e., loss of reflex bladder contractions), which may explainwhy only the excitatory pudendal-to-bladder reflex could be observed inacute SCI cats (Boggs J W, et al., J Neurophysiol 2005; 93:2688-97).

A recent study (Boggs J W, Wenzel B J, Gustafson K J, et al. Bladderemptying by intermittent electrical stimulation of the pudendal nerve. JNeural Eng 2006; 3:43-51) also showed that intermittent pudendal nervestimulation at 33 Hz induced a voiding reflex in cats with an intactspinal cord. However, it is difficult to attribute this voiding solelyto a spinal reflex in spinal intact cats, since the spinobulbospinalmicturition reflex is intact and the PMC coordinates voiding once thebladder contraction is initiated by pudendal nerve stimulation. Inaddition pudendal nerve stimulation evokes a long latency reflexdischarge on bladder postganglionic nerves in spinal intact cats, whichis presumably mediated by a spinobulbospinal pathway because it iseliminated in chronic SCI cats. Therefore, an efficient voiding reflexis expected to be induced in spinal intact cats by pudendal nervestimulation just like the voiding reflex induced by bladder distensionin spinal intact cats (see FIGS. 3A and 5).

EUS Contractions Induced by 20 Hz Pudendal Nerve Stimulation

The urethral outlet resistance in the chronic SCI cats could begenerated by three different mechanisms of EUS activation. The first oneis due to the direct activation of the pudendal efferent input to theEUS by the 20 Hz pudendal nerve stimulation. This could cause a strongEUS contraction that blocks voiding even during a large amplitudebladder contraction (see FIG. 7). The second type of EUS contraction isdue to the excitatory bladder-to-pudendal spinal reflex (i.e., detrusorsphincter dyssynergia). This spinal reflex not only contributes to thelow voiding efficiency in the chronic SCI cats induced by bladderdistension (see FIGS. 3B and 5), but also plays a role in inducing EUScontractions during the bladder contractions induced by 20 Hz pudendalnerve stimulation. The third type of EUS contraction is due to theexcitatory pudendal-to-pudendal spinal reflex. This spinal reflex whichis distributed bilaterally could cause EUS contractions via thecontralateral pudendal nerve even when the ipsilateral pudendal nerve isblocked. Compared to ipsilateral block, bilateral block of the pudendalnerves further increased voiding efficiency to a level that is notsignificantly different from spinal intact cats (see FIG. 5). This showsthat the EUS contraction was partially induced by reflex efferentactivity in the contralateral pudendal nerve. Due to the three types ofEUS contractions induced during 20 Hz pudendal nerve stimulation,voiding efficiency was maximal during simultaneous block of the pudendalnerves bilaterally (see FIG. 5).

A previous study (Sawan M, Hassouna M M, Li J S, et al. Stimulatordesign and subsequent stimulation parameter optimization for controllingmicturition and reducing urethral resistance. IEEE Trans Rehabil Eng1996; 4:39-46) in chronic SCI dogs claimed that complete bladderemptying could be achieved without dorsal rhizotomy by stimulating thesacral spinal roots at frequencies of 300-350 Hz to fatigue the EUS.However, another study (Ishigooka M, Hashimoto T, Sasagawa I, et al.Modulation of the urethral pressure by high-frequency block stimulationin dogs. Eur Urol 1994; 25:334-7) showed that pudendal nerve stimulationbetween 100 and 1,000 Hz could only fatigue the EUS by 30-45% since themajority of the EUS muscles are slow twitch fibers that are fatigueresistant. Further, fatigue stimulation becomes gradually less effectivefor long-term use since the sphincter could change to become morefatigue resistant (Schmidt R A. Neural prostheses and bladder control.IEEE Eng Med Biol Mag 1983; 2:31-4).

Safety of the High-Frequency Blocking Stimulation

Although the effectiveness of biphasic, high-frequency (10 kHz),charge-balanced, electrical stimulation of the pudendal nerves has beendemonstrated in this and previous studies, (Tai C, et al., J Urol 2004;172:2069-72 and Tai C, et al., J Urol 2005; 174:782-6) it remains to bedetermined if this stimulation is safe for long-term use. It is knownthat biphasic, charge-balanced, electrical pulses will cause less damageto the nervous tissue than uniphasic pulses (Agnew W F, McCreery D B.Neural prostheses: Fundamental studies. Englewood Cliffs, N.J.:Prentice-Hall; 1990), but the long-term use of this nerve blockingmethod needs to be evaluated in animal studies before testing in humans.Acute damage of the pudendal nerve caused by high-frequency biphasicstimulation seems unlikely since our previous study (Tai C, et al., JUrol 2005; 174:782-6) in animals showed that this blocking stimulationapplied repetitively (1 min stimulation every 1-3 min) during a periodof 43 min did not alter the neurally evoked EUS response. The potentialhuman application of the high-frequency nerve blocking method will belimited to 3-5 times a day for 1-3 min each time to induce voiding inSCI people. The risk of nervous tissue damage is low when the nerve isonly stimulated for a short time during 24 hr (Agnew W F, McCreery D B.Neural prostheses: Fundamental studies. Englewood Cliffs, N.J.:Prentice-Hall; 1990).

Human Application

Although intermittent voiding responses in quadruped animals isassociated with squirting of urine and territorial marking, which isdifferent from voiding in humans that occurs as a steady stream ofurine, intraurethral electrical stimulation at a frequency of 20 Hzexcited the bladder in people with complete SCI (Gustafson K J, CreaseyG H, Grill W M. A catheter based method to activate urethral sensorynerve fibers. J Urol 2003; 170:126-9 and Gustafson K J, Creasey G H,Grill W M. A urethral afferent mediated excitatory bladder reflex existsin humans. Neurosci Lett 2004; 360:9-12). Since the urethra isinnervated by the pudendal nerve, 20 Hz stimulation has been shown to beeffective in activating the bladder in humans (See, e.g., U.S. Pat. No.7,047,078). Inducing a voiding reflex after SCI by pudendal nervestimulation with simultaneous blockage would not require sacralposterior root rhizotomy, thereby improving on the Brindley's method bypreserving the spinal reflexes for bowel, bladder, and sexual functionsin people with SCI. Meanwhile, eliminating the requirement of sacralposterior root rhizotomy also provides hope for people with SCI tobenefit from any advance in neural regeneration and repair techniques inthe future.

The spinal surgery that is needed in Brindley's method to access thespinal roots would not be necessary either, because only the pudendalnerves would be exposed to implant stimulating electrodes. Compared tospinal surgery, the pudendal nerves can be more easily accessed withminimal surgery (see, e.g., Schmidt R A. Technique of pudendal nervelocalization for block and stimulation. J Urol 1989; 142:1528-31 andSpinelli M, Malaguti S, Giardiello G, et al. A new minimally invasiveprocedure for pudendal nerve stimulation to treat neurogenic bladder:Description of the method and preliminary data. Neurourol Urodyn 2005;24:305-9).

Conclusions

In summary, our studies have revealed that significant differences existbetween the spinal micturition reflex in chronic SCI cats and thespinobulbospinal micturition reflex in spinal intact cats. In additionvoiding is very inefficient in chronic SCI cats as in SCI people. Theimprovement in voiding induced by pudendal nerve stimulation and blockin chronic SCI cats provided further evidence indicating the feasibilityof a new neuroprosthetic device to restore micturition function inpeople after SCI.

Example 2

In this study in chronic paraplegic cats we evaluated an alternativemethod to regulate bladder function involving electrical stimulation ofsomatic afferent nerves innervating the perigenital region.

The perigenital skin area is innervated by the pudendal nerve.Electrical stimulation of the pudendal nerve at low frequencies (1-10Hz) (Tai C, Smerin S E, de Groat W C, Roppolo J R. Pudendal-to-bladderreflex in chronic spinal-cord-injured cats. Exp Neurol 197: 225-234,2006b and Walter J S, Wheeler, J S, Robinson C J, Wurster R D.Inhibiting the hyperreflexic bladder with electrical stimulation in aspinal animal model. Neurourol Urodynam 12: 241-253, 1993) can inhibitthe bladder in adult chronic SCI cats, but at high frequencies (20-30Hz) can excite the bladder (Tai C, Smerin S E, de Groat W C, Roppolo JR. Pudendal-to-bladder reflex in chronic spinal-cord-injured cats. ExpNeurol 197: 225-234, 2006b). However, the pudendal nerve innervates manyareas in the pelvic region including the urethra, anal canal, anal andurethral sphincters, and skin. Whether electrical stimulation applied tothe perigential skin area can mimic the effect of pudendal nervestimulation and induce both inhibitory and excitatory effects on bladderin the chronic SCI cats is uncertain, since the mechanical perigenitalstimulation is primarily excitatory after SCI (de Groat W C, Araki I,Vizzard M A, Yoshiyama M, Yoshimura N, Sugaya K, Tai C, Roppolo J R.Developmental and injury induced plasticity in the micturition reflexpathway. Behavioural Brain Res. 92: 127-140, 1998 and Tai C, Miscik C L,Ungerer T D, Roppolo J R, de Groat W C. Suppression of bladder reflexactivity in chronic spinal cord injured cats by activation of serotonin5-HT_(1A) receptors. Exp Neurol 199: 427-437, 2006a).

In humans after chronic SCI bladder inhibition can also be elicited byelectrical stimulation of the dorsal penile/clitoral nerve (a branch ofpudendal nerve) using skin electrodes (Andre R, Schmid D M, Curt A,Knapp P A, Schurch B. Afferent fibers of the pudendal nerve modulatesympathetic neurons controlling the bladder neck. Neurourol Urodynam 22:597-601, 2003; Hansen J, Media S, Nohr M, Biering-Sorensen F, SinkjaerT, Rijkhoff N J M. Treatment of neurogenic detrusor overactivity inspinal cord injured patients by conditional electrical stimulation. JUrol 173: 2035-2039, 2005; Kirkham A P S, Shah N C, Knight S L, Shah P JR, Craggs M D. The acute effects of continuous and conditionalneuromodulation on the bladder in spinal cord injury. Spinal Cord 39:420-428, 2001; Previnaire J G, Soler J M, Perrigot M. Is there a placefor pudendal nerve maximal electrical stimulation for the treatment ofdetrusor hyperreflexia in spinal cord injury patients? Spinal Cord 36:100-103, 1998; Previnaire J G, Soler J M, Perrigot M, Boileau G,Delahaye H, Schumacker P, Vanvelcenaher J, Vanhee J L. Short-term effectof pudendal nerve electrical stimulation on detrusor hyperreflexia inspinal cord injury patients: importance of current strength. Paraplegia34: 95-99, 1996; Vodusek D B, Light J K, Libby J M. Detrusor inhibitioninduced by stimulation of pudendal nerve afferents. Neurourol Urodynam5: 381-389, 1986; and Wheeler J S, Walter J S, Zaszczurynski P J.Bladder inhibition by penile nerve stimulation in spinal cord injurypatients. J Urol 147:100-103, 1992), indicating an inhibitoryperigenital-to-bladder spinal reflex might also exist in adult chronicSCI cats. An effective, non-invasive method that is able to eitherinhibit or induce bladder activity would improve the current clinicalmanagement of the bladder function after SCI. The possibility of usingelectrical stimulation of the perigential skin area to activate theinhibitory perigenital-to-bladder reflex at one frequency, but toactivate the re-emerged excitatory perigenital-to-bladder reflex atanother frequency was investigated in this study in adult chronic SCIcats.

In order to eliminate possible effects of anesthesia on theperigenital-to-bladder reflexes which may influence voiding efficiency,experiments were performed under awake conditions so that the resultswould be directly comparable to the voiding in neonates. This conditionwould also produce results more relevant to the clinical situation.

Methods Spinal Cord Transection and Animal Care

Four female adult cats (2.8-3.4 kg) were spinalized under isofluraneanesthesia (2-3% in O₂) using aseptic surgical techniques. Afterperforming a dorsal laminectomy at T9-T10 vertebral level, a localanesthetic (lidocaine 1%) was applied to the surface of the spinal cordand then injected into the cord through the dura. The spinal cord wasthen cut completely and a piece of gel foam was placed between the cutends (usually a separation of 2-3 mm). The muscle and skin were suturedand after full recovery from anesthesia the animal was returned to itscage. Following spinal transection, the bladder was emptied daily bymanual expression. If manual expression was not successful, a sterilecatheter (3.5 F) was inserted through the urethra to empty the bladder.Ketaprofen (2 mg/kg s.c., twice a day for 3 days) and antibiotics(Clavamox, 15-20 mg/kg s.c. for 7 days) were given following surgery.Experiments were conducted beginning at least 4-5 weeks following spinalcord transection. The cats were used for multiple experiments at amaximal frequency of twice per week. After each experiment the animalwas given 150 mg/kg of ampicillin subcutaneously. Bladder infectionrarely occurred.

Experimental Setup

A sterile double lumen balloon catheter (7 F) was inserted through theurethra into the bladder of the chronic SCI cats without anesthesia. Theballoon was distended by 2 ml of air and then positioned at the bladderneck by gently pulling the catheter back. The balloon prevented leakageof the fluid from the bladder. One lumen of the catheter was connectedto a pump to infuse the bladder with sterile saline at a rate of 2ml/min, and the other lumen was connected to a pressure transducer tomeasure the pressure change in the bladder. As shown in FIG. 9, a pairof sterilized hook electrodes (made from 23G needles) was attached tothe skin (about 1 mm penetration into the skin with 2-4 mm contact) onthe left and right sides of the vagina approximately 1-1.5 cm from thevaginal opening. A piece of medical tape was applied between theelectrode tip and the exposed length of the electrode in order to fixthe electrode in place. The electrodes were soldered to a pair of wiresthat were connected to a stimulator to deliver electrical stimulation.Due to the complete spinal transection, the animals did not sense eitherbladder catheterization or electrical stimulation. During the experiment(usually 4-5 hours) the animals rested comfortably in a padded animaltransport carrier. Since the animal was free to move in the carrier,bladder pressure recordings that were disrupted by the animal'smovements were discarded. At the end of the experiment the catheter waswithdrawn and the electrodes were detached.

Some experiments were conducted to evaluate voiding induced byelectrical or mechanical perigenital stimulation. In these experimentsthe bladder was not catheterized. The voided volume was collected usinga funnel. The animal was lying quietly on a table during the voidingtests.

Stimulation Protocol

Uniphasic pulses (0.2 ms pulse width) of different intensities (1-30 V)and frequencies (0.5-50 Hz) were delivered to the perigenital skin areavia the attached electrodes using a stimulator (Grass MedicalInstruments, S88) with a stimulus isolator (Grass Medical Instruments,SIU5).

In the first group of experiments, the bladder was infused with sterilesaline to one of two different volumes: (1) a volume slightly above themicturition threshold to induce large amplitude (greater than 30 cmH₂O),rhythmic reflex bladder contractions (see FIGS. 10A and 11A); or (2) avolume slightly below the micturition threshold so that large amplitude,reflex bladder contractions did not occur (see FIG. 12A). During thelarge amplitude rhythmic bladder contractions, electrical perigenitalstimulation was applied in order to determine the effective stimulationparameters to inhibit the bladder. The stimulation duration was longerthan the period of at least two bladder contractions in order to clearlydemonstrate an inhibitory effect. The effective stimulation parametersto induce bladder contractions were determined when bladder volume wasbelow micturition threshold. A stimulation duration of 20-50 seconds wasused which was longer than the period of the induced bladdercontractions. Different stimulation parameters were tested in a randomorder, but are shown in ascending intensity and/or frequency forclarity.

In the second group of experiments, the most effective stimulationfrequencies identified during isovolumetric recordings (7 Hz forinhibition, but 30 Hz for excitation) were tested during acystometrogram (CMG, see FIG. 13A) which consisted of a slow infusion ofsaline (2 ml/min) starting with an empty bladder to determine functionalbladder capacity and examine bladder reflex activity during filling. Twoor three control CMGs were performed without stimulation to obtain thecontrol values and evaluate the reproducibility. Then, either inhibitoryor excitatory perigenital stimulation was applied during the CMG toevaluate the inhibitory or excitatory effects by measuring the change inbladder capacity. Stimulation and infusion were stopped after occurrenceof the first micturition reflex contraction, which was defined as thefirst large amplitude (greater than 30 cmH2O), long duration (greaterthan 20 seconds) reflex bladder contraction accompanied by hindlimbstepping movements. Previous studies (Tai C, Miscik C L, Ungerer T D,Roppolo J R, de Groat W C. Suppression of bladder reflex activity inchronic spinal cord injured cats by activation of serotonin 5-HT_(1A)receptors. Exp Neurol 199: 427-437, 2006a and Thor K B, Roppolo J R, deGroat W C. Naloxone induced micturition in unanesthetized paraplegiccats. J Urol 129: 202-205, 1983) showed that hindlimb stepping movementwas a useful marker for the occurrence of a micturition reflex in awakechronic SCI cats. Bladder capacity is defined as the volume thresholdfor inducing a micturition reflex during a CMG. A control CMG wasperformed at the end of the test to confirm the recovery of themicturition reflex. The bladder was emptied after each CMG and a 5-10minute rest period was inserted between CMGs to allow the bladderreflexes to recover.

In the third group of experiments, the ability of the excitatoryelectrical perigenital stimulation (30 Hz) to induce bladdercontractions at different bladder volumes was evaluated. Short periods(20-50 seconds) of stimulation were applied during the CMGs at intervalsrepresenting 4-8 ml increments in the infused volume. Similar tests werealso performed using mechanical perigenital stimulation that is known tobe excitatory in chronic SCI cats (Tai C, Miscik C L, Ungerer T D,Roppolo J R, de Groat W C. Suppression of bladder reflex activity inchronic spinal cord injured cats by activation of serotonin 5-HT_(1A)receptors. Exp Neurol 199: 427-437, 2006a). The mechanical perigenitalstimulation was performed by repeatedly light stroking (2-3 times/secondfor 20-50 seconds, stroke length 2-3 cm) the perigenital skin area usinga cotton swab. The bladder contractions induced by electrical ormechanical perigenital stimulation were compared.

Excitatory electrical (30 Hz) and mechanical perigenital stimuli werealso used to induce voiding in the awake chronic SCI cats. Voidinginduced by electrical perigenital stimulation was tested in 3 cats (2times in each cat). The bladder was first infused to its capacity usinga urethral catheter. Then the catheter was withdrawn and several minuteswere allowed for any spontaneous voiding to occur. If no spontaneousvoiding occurred or after it was finished, a short duration (10-20seconds) electrical perigenital stimulation was repeatedly applied toinduce voiding. Stimulation was continued until voiding stopped. Thenthe bladder was emptied by manual expression to measure residual volume.If manual expression failed, a sterile urethral catheter (3.5 F) wasused to empty the bladder. Voiding tests were also performed usingrepeated, short duration (10-20 seconds) mechanical perigenitalstimulation with the overnight residual bladder volume as the initialbladder volume.

Data Analysis

For the analysis of rhythmic bladder activity, the area under bladderpressure curve, the inter-contraction interval (ICI), and the averagebladder contraction amplitude were measured during the electricalstimulation and were normalized to the measurements during the same timeperiod prior to the stimulation. The contraction frequency isrepresented as 1/ICI because ICI was an infinite value when completebladder inhibition occurred. For the bladder contractions induced byelectrical stimulation at a bladder volume below the capacity, the areasunder the induced bladder pressure curves were measured and normalizedto the maximal measurement during each experimental trial. Smallamplitude (5-30 cmH2O), short duration (less than 20 seconds)pre-micturition contractions (PMCs) also occurred during CMGs prior tothe large amplitude micturition contraction in chronic SCI cats (Tai C,Miscik C L, Ungerer T D, Roppolo J R, de Groat W C. Suppression ofbladder reflex activity in chronic spinal cord injured cats byactivation of serotonin 5-HT_(1A) receptors. Exp Neurol 199: 427-437,2006a). The bladder capacity, the volume threshold to induce the firstPMC, the average PMC amplitude, and the number of PMCs per minute weremeasured and normalized to the measurements during the first controlCMG. The variability of control CMGs were evaluated by comparing themeasurements from the repeated control CMGs to the first control CMG.The amplitude of bladder contractions induced by electrical ormechanical perigenital stimulation during a CMG were compared atdifferent bladder volumes that were normalized to the bladder capacityin each experiment, and then grouped into bins representing 10%increments in bladder volume. For the voiding tests, the voidingefficiency was calculated as the voided volume divided by the totalvolume (voided volume+residual volume). Repeated measurements in thesame animal during different experiments were averaged. The normalizeddata from different animals are presented as mean±SEM. One sampleStudent t-test and paired Student t-test were used to detect statisticalsignificance (P<0.05) except in two instances as indicated in the textwhere unpaired Student t-test was used. Linear regression analysis (95%confidence interval) and ANOVA analysis were used to determine whetherthe amplitude of bladder contractions induced by perigenital stimulationincreased as the bladder volume increased.

Results Reflex Bladder Activity in Awake Chronic SCI Cats

In awake chronic SCI cats, infusion of saline into the bladder at a rateof 2 ml/min when the bladder neck was blocked with a balloon catheterproduced an immediate small increase in baseline bladder pressure (3-8cmH₂O) and later three types of reflex bladder activity: (1) lowamplitude (5-30 cmH2O), transient (10-20 second duration) increases inbladder pressure (termed pre-micturition contractions—PMCs, see FIG.13A) that occurred in the absence of hindlimb movements, (2) largeamplitude (30-100 cmH₂O, 20-100 second duration) increases in bladderpressure (micturition contractions, see FIG. 13A) that were accompaniedby rhythmic alternating movements of the hindlimbs resembling steppingmovements, and large amplitude rhythmic isovolumetric contractions(1-3/min, 30-100 cmH₂O in amplitude, 20-100 second duration, see FIG.10A and FIG. 11A) that persisted after the bladder infusion was stoppedat the end of a CMG when the bladder volume was above the micturitionthreshold (20-120 ml) and the first micturition contraction was induced(see FIG. 13A and FIG. 14A). The rhythmic hindlimb movements were alsoelicited by manual compression of the bladder when attempting to expressurine during daily nursing care or during voiding induced by tactilestimulation of the perigenital region. The association of somaticreflexes with voiding has also been reported in previous studies (Tai C,Miscik C L, Ungerer T D, Roppolo J R, de Groat W C. Suppression ofbladder reflex activity in chronic spinal cord injured cats byactivation of serotonin 5-HT 1A receptors. Exp Neurol 199: 427-437, 2006and Thor K B, Roppolo J R, de Groat W C. Naloxone induced micturition inunanesthetized paraplegic cats. J Urol 129: 202-205, 1983) and is auseful marker for the occurrence of a micturition reflex. Because someexperiments were performed with the urethral outlet closed whichprevents elimination of the bladder contents, the simultaneousoccurrence of a large amplitude bladder contraction and hindlimbmovements was used as an indicator of the first micturition reflexduring the CMG (see FIG. 13A and FIG. 14A). The cystometric parameterswere relatively constant in the same animal over the course of manyexperiments during a several week period.

2. Inhibitory Perigenital-to-Bladder Spinal Reflex

The inhibitory effect of electrical perigenital stimulation on largeamplitude, rhythmic reflex bladder activity in awake chronic SCI catswas dependent on stimulation frequency (0.5-50 Hz, FIG. 10A). Atstimulation frequencies of 5 Hz and 7 Hz, the electrical perigenitalstimulation significantly (P<0.05) reduced the area under the bladdercontraction curves (70-80%, FIG. 10B) and decreased the frequency(1/ICI) of the rhythmic bladder contractions (70-80%, FIG. 10C), but notthe average contraction amplitude (P>0.05, FIG. 10D) compared to thebladder activity prior to the stimulation.

The inhibition of large amplitude, rhythmic reflex bladder activity at astimulation frequency of 7 Hz was also dependent on stimulationintensity (FIG. 11A). At an intensity above 4 V, the stimulationsignificantly (P<0.05) reduced the area under the bladder contractioncurves (70%, FIG. 11B). It also significantly (P<0.05) decreased thefrequency of the rhythmic bladder contractions at an intensity above 9 V(60%, FIG. 11C), but the average contraction amplitude was not decreasedsignificantly at any stimulation intensity (P>0.05, FIG. 11D).

3. Excitatory Perigential-to-Bladder Spinal Reflex

Although a bladder excitatory effect induced by electrical perigenitalstimulation could not be observed during the large rhythmic bladdercontractions (FIGS. 10 and 11), it became obvious when bladder volumewas below micturition threshold and the large rhythmic bladdercontractions were absent (FIGS. 12A and 12B). The excitatoryperigenital-to-bladder reflex also depended upon the electricalstimulation frequency and intensity. Large amplitude (greater than 30cmH₂O), long duration (greater than 20 sec) bladder contractions wereinduced by stimulation at frequencies between 20 Hz and 40 Hz (FIG.12A). This excitatory effect was diminished as the stimulation intensitydecreased (FIG. 12B). Electrical stimulation at 30 Hz produced bladdercontractions significantly (P<0.05) larger than those produced atfrequencies of 10 Hz or 50 Hz (FIG. 12C). The threshold intensity wasabout 5 V, but at least 11 V was required in order for a 30 Hzstimulation to induce a maximal bladder contraction (FIG. 12D).

4. Micturition Volume Threshold Modulated by Perigenital Stimulation

The threshold bladder volume (i.e. bladder capacity) to induce amicturition reflex contraction was significantly (P<0.05) increased35±13% above control capacity by electrical perigenital stimulation atthe optimal frequency (7 Hz) for inhibiting rhythmic contractions (FIGS.13A and 13B). Stimulation at this frequency also significantly (P<0.05)increased (187±42% above control value) the bladder volume necessary toinduce the first PMC (FIGS. 13A and 13C). However, the average amplitudeand the frequency of PMCs (FIGS. 13D and 13E) and the amplitude of thefirst micturition contraction (see FIG. 13A) were not significantly(P>0.05) changed by the stimulation. Conversely, electrical perigenitalstimulation at the optimal frequency (30 Hz) for inducing bladderexcitation significantly (P<0.05) reduced bladder capacity 21±3% belowthe control capacity (FIGS. 14A and 14B).

5. Influence of Bladder Volume on the Excitatory Perigenital-to-BladderSpinal Reflex

The reflex bladder contractions evoked by electrical or mechanicalstimulation of the perigenital region were elicited at different timesduring bladder filling to evaluate the influence of bladder volume onthe reflex response. As shown in FIG. 15A when short periods (20seconds) of electrical perigenital stimulation (15 V, 30 Hz) wererepeatedly applied after 4 ml increments in bladder volume during a CMG,the stimulation induced large amplitude (greater than 30 cmH₂O)relatively consistent bladder contractions over a range of bladdervolumes. Similarly, mechanical perigenital stimulation (repeatedlystroking the perigenital skin with a cotton swab at a rate of 2-3times/second) could also induce large bladder contractions of relativelyconsistent amplitude at increasing bladder volumes (FIG. 15B). Theaverage amplitude of the bladder contractions induced by eitherelectrical (30 Hz) or mechanical stimulation (FIGS. 15C and 15D) werenot significantly influenced by the bladder volume (P>0.05, ANOVAanalysis and linear regression analysis, N=3).

6. Voiding Induced by Perigenital Stimulation

In awake chronic SCI cats continuous electrical (30 Hz) or mechanicalperigenital stimulation released fluid from the bladder. However, thevoiding was inefficient probably due to detrusor sphincter dyssynergia,and usually only a small amount of bladder volume was released at theend of stimulation (i.e. a post-stimulus voiding). Therefore, shortperiods (10-20 seconds) of electrical or mechanical perigentialstimulation were repeatedly applied (3-6 second interval). This releasedsmall amounts of fluid (1-10 ml) at the end of every period ofstimulation. Usually 5-10 periods of stimulation over 1-5 minutes weresufficient to elicit the maximal bladder emptying.

Voiding induced by mechanical perigenital stimulation was tested usingthe overnight residual volume as the initial bladder volume. Totalvoided volume and the residual volume were measured in 3 cats over a 2month period (FIG. 16A) together with the average amplitude ofmicturition contractions induced by bladder distension or by mechanicalperigenital stimulation (FIG. 16B). The daily residual bladder volumesdetected by manual expression or by emptying using a urethral catheterwere relatively large ranging from 20-80 ml (see FIG. 16A). Meanwhile,frequent incontinent episodes resulting in the release of small amountof urine were also observed in these animals. Similar spontaneousbladder activity was also reported in a recent study (Pikov V, BullaraL, McCreery D B. Intraspinal stimulation for bladder voiding in catsbefore and after chronic spinal cord injury. J Neural Eng 4: 356-368,2007). Voiding efficiencies induced by mechanical perigenitalstimulation in these cats were very different (cat #1: 6.7±3.2%, cat #2:43.2±7.7%, cat #3: 95.0±3.3%) with an average voiding efficiency of48.3±25.6% in the 3 animals (30-32 tests per cat). This may be due tothe fact that mechanical perigenital stimulation generated the smallestbladder pressure (38.8±2.5 cmH₂O) in cat #1, while it induced thelargest bladder pressure (88.1±8.8 cmH₂O) in cat #3 (FIG. 16B). In cat#1 the bladder pressure induced by mechanical perigenital stimulationwas significantly (P<0.05, unpaired t-test) smaller than the bladderpressure (64.7±4.8 cmH₂O) during the distention-induced micturitionreflex (see FIG. 16B). It is also noteworthy that the efficiency ofmechanical perigenital stimulation to induce voiding changed in the sameanimal over time (FIG. 16A).

Voiding induced by electrical stimulation was performed by firstinfusing the bladder to the capacity. After withdrawing the urethralcatheter spontaneous voiding only occurred in one cat (cat #2) with avoiding efficiency ranging from 60% to 70% (2 tests). No spontaneousvoiding was observed in the other 2 cats (cats #1 and #3). Repeated,short duration electrical perigenital stimulation (30 Hz, 30 V, 0.2 ms)was then tested to empty the bladder. The initial bladder volumes rangedfrom 21 ml to 116 ml. Voiding efficiency ranged from 30% to 100% with anaverage of 83.3±10.1% (N=3), that was not correlated with the initialvolume or the voiding efficiency induced by mechanical perigenitalstimulation in each cat. There was no significant difference (P<0.05)between the voiding efficiencies induced by mechanical or electricalperigenital stimulation.

7. Comparison Between Electrical and Mechanical Perigential Stimulation

Under isovolumetric conditions with bladder volume below the micturitionthreshold, electrical stimulation (30 Hz, 20-30 V) induced significantlylarger bladder contractions (P<0.05, unpaired t-test) compared to thoseinduced by mechanical stimulation in cats #1 and #3, but not in cat #2(FIG. 17). Electrical stimulation produced post-stimulus voiding in allcats tested and generated isovolumetric bladder contraction pressuresgreater than 50 cmH₂O (FIG. 17). But the mechanical stimulationgenerated isovolumetric bladder contraction pressures less than 50 cmH₂Oin cat #1 (FIG. 17) and often failed to induce post-stimulus voiding inthis cat (FIG. 16A).

At the bladder volumes below micturition threshold the 30 Hz electricalstimulation induced isovolumetric bladder contractions that lasted42.2±3.9 sec on average (FIG. 18A), but the mechanical stimulation couldinduce isovolumetric bladder contractions that were maintained for aslong as the stimulation was continued (FIG. 18B). The maximal mechanicalstimulation duration tested was about 3 minutes. At the bladder volumesabove micturition threshold when large rhythmic bladder contractionswere occurring, electrical stimulation at 7 Hz inhibited bladder (FIG.18C), but mechanical stimulation slightly facilitated the bladdercontractions evidenced by a longer contraction duration compared to thebladder activity prior to the stimulation (FIG. 18D). However, thisfacilitation was not observed during 30 Hz electrical stimulation (seeFIG. 17A). Neither mechanical nor 30 Hz electrical stimuli significantlyincreased the amplitudes of the rhythmic bladder contractions (see FIG.18D and FIG. 17A).

Discussion

This study revealed that in awake chronic SCI cats electricalstimulation of afferent nerves in the perigenital area could eliciteither an inhibitory or an excitatory effect on the bladder depending onthe frequency of stimulation. The inhibitory effect was maximal at astimulation frequency of 5-7 Hz (FIG. 10 and FIG. 13), whereas theexcitatory effect was maximal at 30 Hz (FIG. 12 and FIG. 14). Both theexcitatory electrical perigenital stimulation (30 Hz) and the mechanicalperigenital stimulation induced large bladder contractions that were notdependent on bladder volume (FIG. 15). Post-stimulus voiding was alsoinduced by the two types of excitatory perigenital stimuli (FIG. 16).However, the properties of the perigenital-to-bladder reflex induced byelectrical or mechanical stimulation were significantly different (seeFIGS. 17 and 18) indicating that either different afferent pathways wereactivated or that the same afferent pathways were activated by these twostimuli in very different ways.

Mechanical stimulation only induced an excitatory effect on bladder, butelectrical stimulation could induce either an excitatory or aninhibitory effect (see FIG. 18). The excitatory effect induced bymechanical stimulation lasted as long as the stimulation continued, butthe electrical stimulation only induced short duration bladdercontractions (FIG. 18A-B). These differences may be caused by the factthat afferent nerves are activated asynchronously by mechanicalstimulation, but they are activated synchronously by each electricalpulse. The frequency of afferent firing can be selected by electricalstimulation, but mechanical stimulation may activate afferent nerveswith a large range of firing frequencies. Electrical stimulation couldactivate both mechano- and non-mechano-sensitive afferent nerves fromboth skin and muscles, while mechanical stimulation mainly activates themechano-sensitive afferent nerves from the skin.

Similar to the mechanical stimulation, the excitatory electricalstimulation also induced large amplitude bladder contractions at bothlow and high bladder volumes (see FIG. 15), indicating that theexcitatory perigenital-to-bladder reflexes are independent of afferentinput from the bladder. Electrical stimulation at 30 Hz probablyactivated the re-emerged, excitatory perigenital-to-bladder spinalreflex in chronic SCI cats, indicating that the average firing frequencyof the afferent nerves activated by mechanical stimulation might be 30Hz. A previous study (Kawatani M, Tanowitz M, de Groat W C.Morphological and electrophysiological analysis of the peripheral andcentral afferent pathways from the clitoris of the cat. Brain Res 646:26-36, 1994) showed that light constant pressure on the clitoris of catproduced sustained afferent firing of pudendal nerve pathway with amaximal frequency of 40 Hz. In addition to directly activating theexcitatory perigenital-to-bladder spinal reflex (FIG. 12 and FIG. 15A),the electrical stimulation could also facilitate the micturition reflexinduced by bladder distension and reduce the micturition volumethreshold during CMG (FIG. 14). The direct excitatory effect on thebladder lasted less than a minute (see FIG. 18A), but the facilitatoryeffect during CMGs could persist for many minutes (see FIG. 14). Thus,it is possible that the 30 Hz electrical stimulation induces bothexcitatory and inhibitory effects. The excitatory effect may be dominantinitially but then suppressed by an inhibitory effect that turns off theperigenital-to-bladder reflex. However, the facilitatory effect on themicturition reflex pathway induced by bladder distention seems to beinsensitive to the later inhibition. This suggests that different spinalinterneuronal pathways or mechanisms may be involved in the directexcitatory perigenital-to-bladder reflex and the facilitatory effect onthe bladder-to-bladder reflex.

At the same stimulation intensity, the electrical perigenitalstimulation is inhibitory to the bladder at 5-7 Hz (FIG. 10), butbecomes excitatory at 20-40 Hz (FIG. 12). This raises the possibilitythat the same population of afferent nerves can induce differentresponses at different frequencies or that different populations ofafferent nerves with the same electrical threshold can produceinhibition and excitation. The frequency selection ofperigenital-to-bladder spinal reflexes must occur in the spinal cord.One possible explanation is that the afferent firing at differentfrequencies triggers the release of different neurotransmitters at thefirst order synapses between the afferent neurons and the spinalinterneurons resulting in either an inhibitory or an excitatory effecton the bladder activity. Another possible explanation is that the spinalinterneuronal networks for bladder inhibition or excitation areoptimally tuned at different frequencies. Afferent firing at 30 Hz isoptimally transmitted through the excitatory spinal neural network, but5-7 Hz is optimal for the inhibitory network (de Groat W C, Ryall R W.Reflexex to sacral parasympathetic neurons concerned with micturition inthe cat. J Physiol 200: 87-108, 1969). The idea of frequency tuning ofspinal neural networks involved in bladder function is also evidenced byprevious studies in cats (de Groat W C. Nervous control of the urinarybladder of the cat. Brain Res 87: 201-211, 1975; de Groat W C, Lalley PM. Reflex firing in the lumbar sympathetic outflow to activation ofvesical afferent fibers. J Physiol 226: 289-309, 1972; de Groat W C,Ryall R W. Recurrent inhibition in sacral parasympathetic pathways tothe bladder. J Physiol 196: 579-591, 1968; Fall M, Lindstrom S.Electrical stimulation: A physiologic approach to the treatment ofurinary incontinence. Urol Clin North Am 18: 393-407, 1991 and LindstromS, Fall M, Carlsson C A, Erlandson B E. The neurophysiological basis ofbladder inhibition in response to intravaginal electrical stimulation. JUrol 129: 405-410, 1983). The maximal inhibition via the hypogastricnerve could be obtained when the pudendal afferent pathway wasstimulated at 5 Hz, whereas the spinal inhibition via pelvic nerve couldbe optimally activated at frequencies 5-10 Hz (Fall M, Lindstrom S.Electrical stimulation: A physiologic approach to the treatment ofurinary incontinence. Urol Clin North Am 18: 393-407, 1991 and LindstromS, Fall M, Carlsson C A, Erlandson B E. The neurophysiological basis ofbladder inhibition in response to intravaginal electrical stimulation. JUrol 129: 405-410, 1983). Recurrent inhibition in sacral parasympatheticpathways to the bladder was optimally tuned at frequencies 15-40 Hz (deGroat W C. Mechanisms underlying recurrent inhibition in the sacralparasympathetic outflow to the urinary bladder. J Physiol 257: 503-513,1976 and de Groat W C, Ryall R W. Recurrent inhibition in sacralparasympathetic pathways to the bladder. J Physiol 196: 579-591, 1968).And the pelvic-to-hypogastric reflex was maximally activated at thefrequency of 0.5 Hz (de Groat W C, Lalley P M. Reflex firing in thelumbar sympathetic outflow to activation of vesical afferent fibers. JPhysiol 226: 289-309, 1972).

Although both mechanical and electrical (30 Hz) stimulation inducedlarge bladder contractions at a low bladder volume (FIG. 15), theyfailed to induce voiding during the stimulation in adult chronic SCIcats, probably due to a co-activation of the urethral sphincter—detrusorsphincter dyssynergia (DSD). However, a small amount of fluid releaseoccurred at the end of each short period of stimulation (i.e.post-stimulus voiding, see also, Example 5, below) presumably due to theability of the urethral sphincter striated muscle to relax faster thanthe smooth muscle of the bladder after the stimulation (Tai C, Smerin SE, de Groat W C, Roppolo J R. Pudendal-to-bladder reflex in chronicspinal-cord-injured cats. Exp Neurol 197: 225-234, 2006b). Thepost-stimulus voiding required a high bladder pressure (greater than 50cmH₂O, see FIGS. 16 and 17). The higher the bladder pressure induced bymechanical stimulation, the higher the voiding efficiency that wasproduced (see FIG. 16). However, high pressure voiding is not clinicallyuseful in patients with strong DSD because it could cause autonomicdysreflexia and kidney problems in long-term application (Consortium forSpinal Cord Medicine. Bladder management for adults with spinal cordinjury: a clinical practice guideline for health-care providers. 2006and Vaidyanathan S, Soni B M, Singh G, Oo T, Hughes P L, Mansour P.Flawed trail of micturition in cervical spinal cord injury patients:guidelines for trial of voiding in men with tetraplegia. Spinal Cord41:667-672, 2003). In contrast, mechanical stimulation of theperigenital region is utilized by the mother cats to completely emptythe bladder of neonatal kittens indicating that co-activation of theurethral sphincter is probably not induced in the neonates. Similardifferences have also been noted in neonatal and SCI rats (Kruse M N, deGroat W C. Consequences of spinal cord injury during the neonatal periodon micturition reflexes in the rat. Exp Neurol 125: 87-92, 1994).

As indicated, a series of short burst pulses can induce a small amountof urine release at the end of each short burst stimulation, that is,post-stimulus voiding. This method might be used in SCI patients whohave strong DSD. However, this method requires cutting the sensoryspinal roots, so that the DSD reflex can be eliminated in SCI patients.For SCI patients with strong DSD, perigenital or perianal stimulationmight not be appropriate, requiring treatment with a 3-channelimplantable device with 5-10 kHz bilateral pudendal nerve blockage.

The urological disorders in SCI patients are largely variable dependingon the level of spinal cord injury, complete or incomplete, etc. SomeSCI patients have only a detrusor hyperreflexia (DH, also calleddetrusor overactivity) during storage phase, but no DSD. Some have onlyDSD but no DH. The most devastating case is where SCI patients have bothDH and DSD. The perigenital or perianal stimulation methods can be usedto treat patients without DSD or with lesser DSD, where high bladderpressure will not be generated. The reason for generating high bladderpressure is not due to bladder contraction alone. It is due to bladdercontacting against the closed urethra (such as in the case of DSD). Assuch, perigenital stimulation does not necessarily generate highpressure voiding.

Since the skin and muscles around the perigenital area are innervated bypudendal nerve, the afferent limb of the perigenital-to-bladder reflexis probably in the pudendal nerve. It has been suggested thatstimulation of pudendal afferents could inhibit bladder activity viaactivation of hypogastric nerve when bladder volume was low, and viaspinal inhibition of the parasympathetic efferent pathways to thebladder when bladder volume was high (Fall M, Erlandson B E, Carlsson CA, Lindstrom S. The effect of intravaginal electrical stimulation on thefeline urethra and urinary bladder: neuronal mechanisms. Scand. J UrolNephrol 44(suppl.): 19-30, 1978 and Fall M, Lindstrom S. Electricalstimulation: A physiologic approach to the treatment of urinaryincontinence. Urol Clin North Am 18: 393-407, 1991). A previous study(Tai C, Smerin S E, de Groat W C, Roppolo J R. Pudendal-to-bladderreflex in chronic spinal-cord-injured cats. Exp Neurol 197: 225-234,2006b) in anesthetized chronic SCI cats also showed the involvement ofboth hypogastric and pelvic efferent nerves in the inhibitorypudendal-to-bladder spinal reflex. The inhibitory perigenital-to-bladderreflex demonstrated in this study in awake chronic SCI cats mightinvolve the same efferent pathways (hypogastric and pelvic) as revealedpreviously.

The efferent pathway in the excitatory perigenital-to-bladder reflexmust involve the pelvic nerve. However, whether the facilitatory effectof perigenital afferent input on micturition reflex involves both pelvicand hypogastric nerves is still uncertain. Perigenital stimulation couldeither enhance the pelvic nerve efferent activity or suppress thehypogastric nerve efferent activity to facilitate the micturitionreflex. Further studies are needed to identify the efferent pathwaysinvolved in the facilitation of micturition reflex induced byperigenital afferent activation.

Somato-Visceral Interactions

When the excitatory electrical (30 Hz) or mechanical perigenitalstimulation (somatic) induced large amplitude (greater than 30 cmH₂O)bladder contractions at different bladder volumes (see FIG. 15), thestimulation also triggered bilateral hindlimb stepping movements. Thestepping movements also occurred during the large amplitude (greaterthan 30 cmH2O), long duration (greater than 20 seconds),distention-induced micturition reflexes, but not during the small PMCs(FIGS. 13 and 14), indicating that they were closely associated with theactivation of the micturition reflex and that the stepping movementswere not directly activated by pudendal afferent input independent ofbladder activity. This idea is further supported by the fact that theexcitatory 30 Hz electrical perigenital stimulation did not induce thehindlimb movements during CMGs until a micturition reflex occurred (seeFIG. 14). The hinblimb stepping movement was a useful marker to indicatethe occurrence of a micturition reflex in awake chronic SCI cats (Tai C,Miscik C L, Ungerer T D, Roppolo J R, de Groat W C. Suppression ofbladder reflex activity in chronic spinal cord injured cats byactivation of serotonin 5-HT_(1A) receptors. Exp Neurol 199: 427-437,2006a and Thor K B, Roppolo J R, de Groat W C. Naloxone inducedmicturition in unanesthetized paraplegic cats. J Urol 129: 202-205,1983). This observation also indicates that a full bladder may be one ofthe mechanisms that triggers the occurrence of leg spasticity in SCIhumans.

The perigenital-to-bladder reflex induced by mechanical stimulationexhibited significant plasticity after SCI (Table 1). The excitatoryperigenital-to-bladder reflex, which exists in neonatal kittens,disappears during development and is replaced by an inhibitoryperigenital-to-bladder reflex in adult cats. After SCI the mechanicalstimulation-induced, excitatory perigenital-to-bladder reflex re-emerges(see FIGS. 15-18) and the inhibitory reflex disappears (de Groat W C.Nervous control of the urinary bladder of the cat. Brain Res 87:201-211, 1975; de Groat W C, Araki I, Vizzard M A, Yoshiyama M,Yoshimura N, Sugaya K, Tai C, Roppolo J R. Developmental and injuryinduced plasticity in the micturition reflex pathway. Behavioural BrainRes. 92: 127-140, 1998; de Groat W C, Ryall R W. Recurrent inhibition insacral parasympathetic pathways to the bladder. J Physiol 196: 579-591,1968; and Kirkham A P S, Shah N C, Knight S L, Shah P J R, Craggs M D.The acute effects of continuous and conditional neuromodulation on thebladder in spinal cord injury. Spinal Cord 39: 420-428, 2001). Thisreflex plasticity indicates a significant re-organization occurring inthe spinal cord after chronic SCI.

The effect of electrical perigenital stimulation on bladder reflexes inawake spinal intact cats has not been determined, making it impossibleto evaluate the plasticity of this reflex after chronic SCI. However, inanesthetized spinal-intact cats intra-vaginal electrical stimulation ata frequency of 5-10 Hz inhibited bladder activity (Fall M, Erlandson BE, Carlsson C A, Lindstrom S. The effect of intravaginal electricalstimulation on the feline urethra and urinary bladder: neuronalmechanisms. Scand J Urol Nephrol 44: 19-30, 1978 and Lindstrom S, FallM, Carlsson C A, Erlandson B E. The neurophysiological basis of bladderinhibition in response to intravaginal electrical stimulation. J Urol129: 405-410, 1983). Furthermore, dorsal penile/clitoris nervestimulation at 10 Hz also inhibited both Aδ-fiber mediated (

Jiang C H, Lindstrom S. Prolonged enhancement of the micturition reflexin the cat by repetitive stimulation of bladder afferents. J Physiol517:599-605, 1999) and C-fiber mediated (Mazieres L, Jiang C H,Lindstrom S. The C fibre reflex of the cat urinary bladder. J Physiol513: 531-541, 1998) pelvic-to-pelvic micturition reflexes in spinalintact cats, indicating that electrical stimulation of the perigenitalskin area at these frequencies might be also inhibitory (see Table 1).An excitatory effect of electrical perigenital stimulation on bladderactivity in spinal intact cats might also occur since pudendal nervestimulation in a range of frequencies (1-40 Hz) induced bladdercontractions (Boggs J W, Wenzel B J, Gustafson K J, Grill W M. Spinalmicturition reflex mediated by afferents in the deep perineal nerve. JNeurophysiol 93: 2688-2697, 2005; Boggs J W, Wenzel B J, Gustafson K J,Grill W M. Frequency-dependent selection of reflexes by pudendalafferents in the cat. J Physiol 577: 115-126, 2006; de Groat W C, RyallR W. Reflex to sacral parasympathetic neurons concerned with micturitionin the cat. J Physiol 200: 87-108, 1969; Mazieres L, Jiang C, LindstromS. Bladder parasympathetic response to electrical stimulation ofurethral afferents in the cat. Neurourol Urodynam 16: 471-472, 1997; andShefchyk S J, Buss R R. Urethral pudendal afferent-evoked bladder andsphincter reflexes in decerebrate and acute spinal cats. Neurosci Lett244:137-140, 1998) (see Table 1). The perigenital-to-bladder reflexinduced by electrical stimulation in spinal intact cats needs to befurther investigated in order to fully understand the neuroplasticity ofthis reflex after SCI.

TABLE 1 Comparison of perigenital-to-bladder reflexes induced bymechanical and electrical stimulation in spinal intact and chronic SCIcats Mechanical stimulation Electrical Stimulation Spinal Intact ChronicSCI Spinal Intact Chronic SCI Inhibition Yes No Yes (5-10 Hz)* Yes (5-7Hz) Excitation No Yes Yes (1-40 Hz)** Yes (20-40 Hz) SCI = Spinal CordInjured; *Intravaginal stimulation; **pudendal nerve stimulation

Our current study in awake chronic SCI animals tested the hypothesisthat frequency dependent activation of the inhibitory or excitatorypudendal-to-bladder spinal reflex could be achieved by applyingelectrical stimulation to the perigenital skin area. A previous study(Walter J S, Wheeler, J S, Robinson C J, Wurster R D. Inhibiting thehyperreflexic bladder with electrical stimulation in a spinal animalmodel. Neurourol Urodynam 12: 241-253, 1993) using restrained, awake,chronic SCI cats only investigated the inhibitory effect on the bladderby inserting fine wire electrodes percutaneously to stimulate thepudendal nerve. Bladder excitation by perineal stimulation was alsoinvestigated previously in spinal dogs (Walter J S, Wheeler J S,Robinson C J, Wurster R D. Surface stimulation techniques for bladdermanagement in the spinal dog. J Urol 141:161-165, 1989). Our studyfurther showed that both inhibitory and excitatory bladder responsescould be induced in chronic SCI cats by electrical perigenitalstimulation at different frequencies. Clinical tests using suprapubictapping and jabbing could only induce bladder contractions in about 50%of the SCI subjects (Cardenas D D, Kelly E, Mayo M E. Manual stimulationof reflex voiding after spinal cord injury. Arch Phys Med Rehabil66:459-462, 1985) and manual anorectal stimulation inhibited bladderactivity (Rodriquez A A, Awad E. Detrusor muscle and sphincter responseto anorectal stimulation in spinal cord injury. Arch Phys Med Rehabil60:269-272, 1979). But these clinical tests activated very differentafferent pathways than perigenital stimulation. Currently, a systematicclinical investigation of the frequency dependent bladder responses toperigenital electrical stimulation in SCI subjects is still notavailable. Although a recent study in chronic SCI cats (Example 5)showed that intraspinal microstimulation might be applicable to restorebladder function after SCI, a non-invasive clinical approach to eitherexcite or inhibit the bladder activity will still be very useful andcould be envisioned based on the results presented in our study.

Example 3—Bladder Inhibition or Excitation by Electrical PerianalStimulation in the Chronic SCI Cat

Example 2 demonstrates that electrical perigenital stimulation couldactivate a branch of the pudendal nerve to induce either an inhibitoryor an excitatory spinal reflex to the bladder at different stimulationfrequencies. Since the pudendal nerve also innervates the muscle andskin in the perianal area, we explored in this study if electricalstimulation applied on the perianal skin area could also activate eitheran inhibitory or an excitatory spinal reflex to the bladder depending onstimulation frequency.

Electrical stimulation applied by ring electrodes located on anal plugcan induce contraction of the pelvic floor muscles and inhibition ofdetrusor overactivity (Godec C, Cass A S, Ayala G F. Bladder inhibitionwith functional electrical stimulation. Urology 1975: 6:663-666). Thistechnique has been recommended for stress and urge urinary incontinence(Godec C, Cass A S, Ayala G F. Electrical stimulation for incontinence;technique, selection and results. Urology 1976: 7:388-397). However,intra-anal stimulation produced some problems. For example, the analplug electrodes had to be removed every 2-3 h to pass flatus. Inaddition, many patients could not use these electrodes because of painand discomfort or because of a stenotic or closed anus due to previousinjury or surgery. In order to provide a solution for these problems,the effect of electrical stimulation of the perianal skin on bladderfunction was investigated (Nakamura M, Sakurai T, Tsujimoto Y, Tada Y.Bladder inhibition by electrical stimulation of the perianal skin. Urol.Int. 1986: 41:62-63). It was found that perianal electrical stimulationelicited a suppression of detrusor activity and caused poststimulationimprovement in frequency, urgency, and incontinence. In this study wefurther characterized the spinal reflexes from the perianal skin area tothe bladder using awake chronic SCI cats.

Methods Spinal Cord Transection

Three female cats (2.8-3.4 kg) were spinalized under isofluraneanesthesia using aseptic surgical techniques. After performing a dorsallaminectomy at T9-T10 vertebral level, a local anesthetic (lidocaine 1%)was applied to the surface of the spinal cord and then injected into thecord through the dura. The spinal cord was then cut completely, and apiece of gel foam was placed between the cut ends (usually a separationof 2-3 mm). The muscle and skin were then sutured. After full recoveryfrom anesthesia the animal was returned to its cage. Following spinaltransection, the bladder was emptied daily by manual expression. Ifmanual expression was not successful, a sterile catheter (3.5 F) wasinserted through the urethra to empty the bladder. Ketaprofen (2 mg/kgtwice a day for 3 days) and antibiotics (Clavamox, 15-20 mg/kg for 7days) were given following surgery. Experiments to determine theproperties of perianal-to-bladder spinal reflex were conducted after atleast 4-5 weeks following spinal cord transaction.

Experimental Setup

A sterile double lumen balloon catheter (7 F) was inserted through theurethra into the bladder of the chronic SCI cats without anesthesia. Theballoon was distended by 2 ml of air and then positioned at the bladderneck by gently pulling the catheter back. The balloon prevented leakageof the fluid from the bladder. One lumen of the catheter was connectedto a pump to infuse the bladder with sterile saline at a rate of 2ml/min, and the other lumen was connected to a pressure transducer tomeasure the pressure change in the bladder. A pair of sterilized hookelectrodes (made from 23G needles) was attached to the skin (about 1 mmpenetration into the skin with 2-4 mm contact) on the left and rightsides of the anus approximately 1-1.5 cm from the anal opening. Due tothe complete spinal transection, the animals did not sense eitherbladder catheterization or electrical stimulation. During the experiment(usually 4-5 hours) the animals rested comfortably in a padded animaltransport carrier. Since the animal was free to move in the carrier,bladder pressure recordings that were disrupted by the animal'smovements were discarded. At the end of the experiment the catheter waswithdrawn and the electrodes were detached. After each experiment theanimal was given 150 mg/kg of ampicillin subcutaneously. Multipleexperiments were repeated on the same animal on different days.

Stimulation Protocol

Uniphasic pulses (0.2 ms pulse width) of different intensities (1-30 V)and frequencies (0.5-50 Hz) were delivered to the perianal skin area viathe attached electrodes using a stimulator (Grass Medical Instruments,S88) with a stimulus isolator (Grass Medical Instruments, SIU5).

In the first group of experiments, the bladder was infused to one of thetwo different volumes: (1) a volume slightly above the micturitionthreshold to induce large amplitude (greater than 25 cm H₂O) rhythmicbladder contractions (see FIG. 19A); or (2) a volume slightly below themicturition threshold so that no large amplitude rhythmic bladdercontractions occurred (see FIG. 21A). During rhythmic bladdercontractions, electrical perianal stimulation was applied in order todetermine the effective stimulation parameters to inhibit the bladder.The stimulation duration was longer than the period of at least 2rhythmic bladder contractions in order to confirm the inhibitory effect.The effective stimulation parameters to induce bladder contractions weredetermined when bladder volume was low and large amplitude rhythmiccontractions were absent. Stimulation duration of 30-50 seconds was usedso that a full bladder contraction response could be induced whichincluded the peak of the contraction and the gradual return of bladderpressure to the baseline.

In the second group of experiments, the most effective stimulationparameters to inhibit the bladder (7 Hz) identified in the first groupof experiments were further tested during a slow infusion of the bladder(i.e., during a cystometrogram-CMG, see FIG. 22A). The CMG was alwaysperformed with an initially empty bladder. First, control CMGs wererepeated 2-3 times without stimulation to obtain the control values andevaluate the reproducibility. Then, inhibitory perianal stimulation wasapplied during the CMG to quantify the inhibitory effect by measuringthe change in bladder volume to induce the first large amplitude reflexcontraction (i.e., bladder capacity). Stimulation and infusion werestopped when the first micturition contraction occurred which wasdefined as the first large amplitude (greater than 25 cm H₂O), longduration (greater than 20 sec) reflex bladder contraction that wasaccompanied by hindlimb stepping movements. Previous studies showed thathindlimb stepping movement was a useful marker for the occurrence of amicturition reflex in awake chronic SCI cats. Bladder capacity isdefined as the bladder volume threshold during a CMG which evokes thefirst micturition contraction. The bladder was emptied after each CMGand a 5-10 minute waiting period was allowed between CMGs for thebladder reflexes to recover.

In the third group of experiments, the ability of the excitatoryelectrical stimulation (30 Hz) to induce bladder contractions atdifferent bladder volumes was further evaluated. A short burst (30-50sec) of stimulus pulses was applied during a CMG after infusion ofsaline in 4-8 ml increments.

Data Analysis

For the rhythmic bladder activity, the area under bladder pressurecurve, the inter-contraction interval (ICI), and the average bladdercontraction amplitude were measured during the electrical stimulationand were normalized to the measurements during the same time periodprior to the stimulation. The contraction frequency is represented as1/ICI because ICI is an infinite value when complete bladder inhibitionoccurs. For the bladder contractions induced by electrical stimulationat a bladder volume below capacity, the areas under the induced bladderpressure curves were measured and normalized to the maximal measurementduring each experimental trial. During CMGs small amplitude (10-25 cmH₂O), short duration (less than 20 sec) pre-micturition contractions(PMCs) occurred prior to the large amplitude micturition contraction inchronic SCI cats. PMCs indicate the bladder overactivity due to chronicSCI that plays a role in frequent incontinence after SCI. For the CMGrecordings, the bladder capacity, the volume threshold to induce thefirst PMC, the amplitude and the number of PMCs per minute were measuredand normalized to the measurements during the first control CMG. For thebladder contractions induced by electrical perianal stimulation atdifferent bladder volumes during a CMG the amplitude of the contractionswere measured. The tested bladder volumes were normalized to the bladdercapacity, and then they were grouped in bins with every 10% increase ofthe bladder volume. The normalized data from different experiments arepresented as mean±SEM. Both one sample Student t-test and paired Studentt-test were used to detect statistical significance (P<0.05). Linearregression analysis (95% confidence interval) and ANOVA analysis wereused to determine whether the amplitude of bladder contractions inducedby perianal stimulation was increased as the bladder volume increased.

Results Inhibitory Perianal-to-Bladder Spinal Reflex

The inhibitory effect of electrical perianal stimulation on rhythmicbladder activity in awake chronic SCI cats was dependent on stimulationfrequency. FIG. 19A shows an example of electrical perianal stimulationat different frequencies (0.5-50 Hz) inhibiting rhythmic bladdercontractions. The different stimulation frequencies were applied in arandom order, but they are shown in ascending order in FIG. 19A forclarity. The inhibitory effect on rhythmic bladder activity was obvious(P<0.05) between 3 Hz and 10 Hz as either a decrease in the contractionamplitude or a reduction in the area under bladder pressure curve (FIGS.19A and 19B). The stimulation significantly (P<0.05) decreased theaverage contraction amplitude during the stimulation at 3-10 Hz (FIG.19C), but the frequency of bladder rhythmic contractions was not changedsignificantly (P>0.05, FIG. 19D).

The inhibitory effect on rhythmic bladder activity was also dependent onstimulation intensity. FIG. 20A shows an example of inhibition ofrhythmic bladder contractions at different intensities (5-30 V) using afrequency of 7 Hz. The different stimulation intensities were applied ina random order, but they are shown in ascending order in FIG. 20A forclarity. At stimulation intensity above 8 V, the electrical perianalstimulation significantly (P<0.05) reduced the area under the bladdercontraction curve during the stimulation compared to the bladderactivity prior to the stimulation (FIG. 20B). The stimulation alsosignificantly (P<0.05) decreased the average contraction amplitudeduring the stimulation at an intensity above 8 V (FIG. 20C), but thefrequency of bladder contractions was not changed significantly (P>0.05,FIG. 20D).

2. Excitatory Perianal-to-Bladder Spinal Reflex

The perianal-to-bladder reflex in awake chronic SCI cats could also beexcitatory depending on the electrical stimulation frequency andintensity. FIGS. 21A-B shows examples of bladder contractions induced byelectrical perianal stimulation at different frequencies and intensitieswhen bladder volume was below its capacity. Large amplitude (greaterthan 25 cm H₂O), long duration (greater than 20 sec) bladdercontractions were induced by stimulation at a frequency between 20 Hzand 50 Hz (FIG. 21A). This excitatory effect was enhanced as thestimulation intensity increased (FIG. 21B). Electrical stimulation at 30Hz was optimal to induce bladder contractions since it produced thelargest area under the bladder contraction curve. The effect of 30 Hzwas significantly (P<0.05) larger than the responses produced at afrequency of 10 Hz (see FIG. 21C). In order for 30 Hz stimulation toinduce a large bladder contraction greater than 75% of the maximalresponse, the required stimulation intensity was above 12 V (FIG. 21D).

3. Micturition Volume Threshold Modulated by Perianal Stimulation

The threshold bladder volume (i.e., bladder capacity) to induce amicturition reflex contraction in awake chronic SCI cats wassignificantly increased by the inhibitory electrical perianalstimulation at 7 Hz. FIG. 22A shows an example of repeated CMGrecordings. Compared to the first control CMG, the 7 Hz stimulationdelayed the occurrence of both the first micturition contraction and thefirst pre-micturition contraction (PMC) (FIG. 19A). The bladder capacitywas significantly (P<0.05) increased to 140±10% of the control capacity(FIG. 22B) and the volume threshold to induce the first PMC wassignificantly (P<0.05) increased to 255±60% of the control value (FIG.22C). The average amplitude and the frequency of the PMCs were alsosignificantly (P<0.05) decreased to 56±3% and 41±5% of the control value(FIG. 22D-E). The amplitude of the first micturition contraction was notinfluenced by the inhibitory 7 Hz stimulation (see FIG. 22A).

4. Independence of Excitatory Perianal-to-Bladder Spinal Reflex onBladder Volume

In addition to inhibiting the bladder-to-bladder micturition reflex, theperianal afferent input could also induce an excitatoryperianal-to-bladder reflex in awake chronic SCI cats, which was notdependent on the bladder volume. FIG. 23A shows a CMG recording where ashort burst (40 second duration) of electrical stimulation (25 V, 30 Hz)was repeatedly applied to the perianal area at regular intervalsfollowing approximately 4 ml increments in bladder volume. Thestimulation induced large (greater than 25 cm H₂O) amplitude bladdercontractions at a variety of bladder volumes. FIG. 23B shows the averageamplitude of bladder contractions induced by electrical (30 Hz)stimulation at different bladder volumes. Both ANOVA analysis and linearregression analysis showed that the amplitude of bladder contractionswas not significantly (P>0.05) increased when the bladder volume wasincreased (FIG. 23B).

Discussion

This study revealed that in awake chronic SCI cats, electricalstimulation of afferent nerves located in the perianal skin area couldelicit either an inhibitory or an excitatory spinal reflex to thebladder depending on the frequency of stimulation. The inhibitory effectwas significant at a stimulation frequency of 3-10 Hz (FIG. 19), but theexcitatory effect was maximal at 30 Hz (FIG. 21C). The inhibitorystimulation at 7 Hz significantly increased bladder capacity andinhibited the pre-micturition contractions (FIG. 22). The excitatoryelectrical (30 Hz) perianal stimulation induced large bladdercontractions that were not dependent on bladder volume (FIG. 23). Theresults obtained in this study are similar to those obtained fromelectrical stimulation of perigenital area, described in Example 2,indicating that the pudendal afferents from both anal and genital areascould have the same modulatory role on bladder activity in chronic SCIcats.

Mechanical anal stimulation could also induce either an inhibitory or anexcitatory effect on bladder activity. Inhibition of bladdercontractions by stretching the anal sphincter was observed in peoplewith or without SCI (Kock N G, Pompeius R. Inhibition of vesical motoractivity induced by anal stimulation. Acta Chir Scand 1963: 126:244-250and Rodriquez A A, Awad E. Detrusor muscle and sphincteric response toanorectal stimulation in spinal cord injury. Arch. Phys. Med. Rehabil.1979: 60:269-272). Excitation of the bladder was also seen in SCIsubjects by light perianal touch, pinprick, introduction of rectalcatheter, or alternatively inflation and deflation of a rectal balloon(Rossier A, Bors E. Detrusor responses to perianal and rectalstimulation in patients with spinal cord injuries. Urol. Int. 1964:18:181-190). It seems that mild stretch or light touch of the anus tendsto be excitatory while vigorous stretch tends to cause inhibition. Inthis study, we further demonstrated that the perianal-to-bladder spinalreflex in awake chronic SCI cats could be either inhibitory orexcitatory depending on the frequency of electrical stimulation.Previous studies (Godec C, et al., Urology 1975: 6:663-666; Godec C, etal., Urology 1976: 7:388-397; and Nakamura M, et al., Urol. Int. 1986:41:62-63) in human SCI subjects using electrical stimulation (20 Hz) ofthe anal area only demonstrated bladder inhibition. Our study indicatedthat using perianal stimulation at different frequencies might alsoinduce bladder excitation in people with SCI. Inducing either inhibitionor excitation using the same stimulation electrode will be very usefulin clinical applications.

Since the skin and muscle around the anal area are innervated bypudendal nerve, the electrical stimulation used in this study must haveactivated a branch or branches of this nerve. The inhibitorypudendal-to-bladder reflex has been demonstrated in spinal intact andacute SCI cats by intra-vaginal electrical stimulation. It was suggestedthat stimulation of pudendal afferents could inhibit bladder activityvia activation of hypogastric nerve when bladder volume was low, and viaspinal inhibition of parasympathetic activity in the pelvic nerve whenbladder volume was high (Fall M, Erlandson B E, Carlsson C A, LindstromS. The effect of intravaginal electrical stimulation on the felineurethra and urinary bladder: neuronal mechanisms. Scand. J. Urol.Nephrol. suppl. 1978: 44:19-30 and Fall M, Lindstrom S. Electricalstimulation: A physiologic approach to the treatment of urinaryincontinence. Urol. Clin. North Amer. 1991: 18:393-407). A previousstudy (Wheeler J S, Walter J S, Zaszczurynski R I. Bladder inhibition bypenile nerve stimulation in spinal cord injury patients. J. Urol. 1992:147:100-103) in anesthetized chronic SCI cats also showed theinvolvement of both hypogastric and pelvic efferent nerves in theinhibitory pudendal-to-bladder spinal reflex. The inhibitoryperianal-to-bladder reflex demonstrated in this study in awake chronicSCI cats might involve the same efferent pathways (i.e., hypogastric andpelvic) as revealed previously. Meanwhile, the electrical perianalstimulation can also activate an excitatory perianal-to-bladder spinalreflex to induce large amplitude bladder contractions at both low andhigh bladder volumes (see FIG. 22), indicating that the excitatoryperianal-to-bladder reflex is independent on the afferent input from thebladder. This suggests that the excitatory afferent input from theperianal area may have projections to the parasympathetic preganglionicneurons via spinal circuitry separate from that activated by the pelvicafferent input from the bladder.

The electrical perianal stimulation is inhibitory to the bladder at 3-10Hz (FIG. 19), but becomes excitatory at 20-50 Hz (FIG. 21). Since at thesame stimulation intensity the same afferent nerve fibers are activated,the frequency selection of the perianal-to-bladder spinal reflex mustoccur in the spinal cord. One possible explanation is that the afferentfiring at different frequencies may trigger the release of differentneurotransmitters at the first spinal synapse between the primaryafferent axons and spinal interneurons resulting in either an inhibitoryor an excitatory effect on the bladder activity. Another possibleexplanation is that the spinal interneuronal networks are optimallytuned at different frequencies for bladder inhibition or excitation.Afferent firing between 20-50 Hz is optimally transmitted through theexcitatory spinal neural network, but 3-10 Hz is optimal for theinhibitory network. Frequency tuning of spinal neural networks is alsoevident in previous studies in cats (Lindstrom S, Fall M, Carlsson C A,Erlandson B E. The neurophysiological basis of bladder inhibition inresponse to intravaginal electrical stimulation. J. Urol. 1983:129:405-410 and Fall M, et al., Urol. Clin. North Amer. 1991:18:393-407). The maximal inhibition via the hypogastric nerve could beobtained when the pudendal afferent pathway was stimulated at 5 Hz,whereas the spinal inhibition via pelvic nerve could be optimallyactivated at frequencies between 5 and 10 Hz (Lindstrom S, et al., J.Urol. 1983: 129:405-410 and Fall M, et al., Urol. Clin. North Amer.1991: 18:393-407).

Our current study in awake, chronic SCI animals tested the hypothesisthat frequency dependent activation of the inhibitory or excitatorypudendal-to-bladder spinal reflex could be induced by applyingelectrical stimulation to the perianal skin area. A previous study(Walter J S, Wheeler J S, Robinson C J, Wurster R D. Inhibiting thehyperreflexic bladder with electrical stimulation in a spinal animalmodel. Neurourol. Urodynam. 1993: 12:241-253) using restrained, awake,chronic SCI cat only investigated the inhibitory effect on bladder byinserting fine wire electrodes percutaneously to stimulate the pudendalnerve. Whether the excitatory bladder response induced in this studycould produce efficient voiding depends on the relaxation of externalurethral sphincter (EUS). Although the EUS activity was not investigatedin this study, the perianal stimulation presumably activated the EUS dueto the excitatory pudendal-to-pudendal spinal reflex. However, thevoiding problem due to dyssynergic EUS contraction could be overcome byinducing post-stimulus voiding (Brindley G S. The first 500 sacralanterior root stimulator implants: general description. Paraplegia 1994:32:795-805). The approach employed in this study in awake SCI cats tocharacterize reflexes from the perianal skin to the urinary bladdermight provide an alternative and practical clinical method to managebladder function after SCI.

Example 4

The experimental setup is shown in FIG. 24. One female and two male cats(5.1 to 6.0 kg) were used under α-chloralose anesthesia (60 mg/kg i.v.,supplemented as needed). The temperature of the animal was maintained at35-37° C. using a heating pad. The ureters were cut and drainedexternally. A catheter (5 F) was inserted through the bladder dome intothe proximal urethra and secured by a ligature near the bladder neck.The catheter was attached to both an infusion pump and a pressuretransducer via a T-connector. The urethra was infused continuously withsaline solution at the rate of 1-2 ml/min. The back pressure in theurethral perfusion system caused by contractions of urethral sphincterwas recorded via the pressure transducer. Pudendal nerves were accessedposteriorly in the sciatic notch and cut bilaterally to eliminate anyeffect of the pudendal-to-pudendal spinal reflex on the experimentresults. A small pool was formed around the pudendal nerve (usually onthe left side) by retracting skin flaps and the pool was filled withmineral oil. A tripolar cuff electrode [shown as Stim. A in FIG. 24,Micro Probe, Inc., NC223(Pt)] was placed around one pudendal nerve(usually on the left side). The electrode leads were made of platinumwires (diameter 0.25 mm) with a distance of 2 mm between the leads. Asecond pair of stainless steel hook electrode (shown as Stim. B in FIG.24) was placed on the pudendal nerve at a location central to thetripolar cuff electrode in order to test whether stimulation A couldblock the urethral response induced by stimulation B. A digitalthermometer was used to monitor the temperature of the small mineral oilpool. The probe of the thermometer was placed adjacent to the cuffelectrode and the pudendal nerve. The temperature of the mineral oilpool was adjusted between 14° C. and 37° C. by manually infusing cold orwarm mineral oil into the small pool. The temperature of the mineral oilpool was maintained in a range of ±0.5° C. during each electricalstimulation period.

The high-frequency blocking stimulation tested in this study wasbiphasic, charge-balanced continuous rectangular wave (see FIG. 24). Ata certain temperature (15, 20, 27, or 37° C.), the high-frequencystimulation (10 seconds duration) was tested at different frequenciesranging from 1 kHz to 10 kHz in 1 kHz increments to search for theminimal blocking frequency. The intensity of the high-frequencystimulation was set at about 1.5 times of the blocking thresholddetermined at temperature of 37° C. The determination of blockingthreshold was fully described in our previous study (Tai C, Roppolo J R,de Groat W C (2005c). Response of external urethral sphincter to highfrequency biphasic electrical stimulation of pudendal nerve. J Urol174:782-7), which involved testing on both intensity and frequency.After the minimal blocking frequency was determined at differenttemperatures, stimulation frequency of 4 kHz was chosen to test for anerve block effect at different temperatures ranging from 14.5° C. to36.5° C. in 2° C. increments. The same test was repeated 3 times in eachanimal, and the data were presented as mean±standard error across allanimals. The high-frequency, biphasic stimulation waveforms (1-10 kHz)were generated by a computer with a digital-to-analog circuit board(National Instruments, AT-AO-10), which was programmed using LabViewprogramming language (National Instruments). Linear stimulus isolators(World Precision Instruments, A395) were used to deliver the biphasicconstant current pulses to the nerve.

Results

As shown in FIG. 24, the blocking stimulation A (Stim. A) was used toblock the urethral pressure responses induced by the excitingstimulation B (Stim. B). When the intensity of high frequency blockingstimulation was above the blocking threshold, the urethral pressureinduced by the high-frequency blocking stimulation alone was graduallyreduced as the stimulation frequency increased (see FIG. 25A). Duringthe 10 second high-frequency stimulation, a frequency high enough tosuppress the urethral pressure response at the end of the stimulation(for example, 8 kHz as shown in FIG. 25A) could also block the pudendalnerve conduction (see FIG. 25B). FIG. 25B shows that the samestimulation (8 kHz, 2 mA) in the same animal blocked the urethralresponses induced by the centrally located stimulation B (see FIG. 24).The urethral pressure responses could not be blocked by stimulation A ifthe stimulation B was moved to a location on the pudendal nerve distalto the stimulation A (see FIG. 25C). This further indicated thatpudendal nerve block rather than urethral muscle fatigue was responsiblefor the loss of the urethral pressure responses during thehigh-frequency stimulation (see FIG. 25A). At stimulation intensity of1.5 times of the blocking threshold as used in this study, FIG. 25A-C,and our previous study (Tai C, et al., J Urol 174:782-7) have shown thatnerve conduction block occurred when the urethral pressure response wascompletely suppressed at the end of 10 second stimulation. Therefore, inthis study the urethral pressure at the end of 10 second stimulation wasmeasured to indicate the blocking effect (see FIG. 27B).

The minimal stimulation frequency required to completely block thepudendal nerve was reduced as the temperature was decreased. FIGS. 26A-Dshows the urethral pressure responses to different frequencies ofblocking stimulation A at different temperatures. At a temperature of37° C. (FIG. 26A), the minimal stimulation frequency required to blockpudendal nerve conduction during the 10 second stimulation was 6 kHz.This minimal frequency was reduced to 5 kHz when the temperature wasdecreased to 27° C. (FIG. 26B). At a temperature of 20° C. (FIG. 26C),the block occurred at the end of the 10 second stimulation withfrequency of 4 kHz. Further decreasing the temperature to 15° C. (FIG.26D) resulted in the block occurring at stimulation frequency of 4 kHzin less than 10 seconds. FIGS. 26A-D shows that the minimal blockingfrequency changed from 6 kHz at 37° C. to 4 kHz at 15-20° C. The testsas shown in FIGS. 26A-D were repeated 3 times in each animal FIG. 27Asummarizes the experimental results from all 3 animals (N=9). Theurethral pressure at the end of 10 second high-frequency stimulation wasmeasured to indicate the blocking effect (see FIG. 27B). At a certaintemperature, the urethral pressures induced by stimulation at differentfrequencies were normalized to the pressure induced by stimulation at 1kHz. As shown in FIG. 27A, the normalized pressure-frequency curve wasshifted toward a lower frequency when the temperature was decreased from37° C. to 15° C. The minimal stimulation frequency to induce a completeblock was reduced from 6 kHz to 4 kHz as the temperature was decreasedfrom 37° C. to 15° C.

Based on the results shown in FIG. 27A stimulation frequency of 4 kHzwas chosen to determine more precisely the temperature threshold atwhich the nerve block occurred. The frequency of 4 kHz was testedbecause the urethral pressure response at this frequency changed from 0%to almost 100% when the temperature was increased from 15° C. to 37° C.(see FIG. 27A). Therefore, testing 4 kHz could show a full range ofresponses.

FIG. 28A shows how the urethral response to the 4 kHz stimulationrecovered gradually when the temperature was increased from 14.5° C. to36.5° C. in 2° C. increments. At temperatures below 24.5° C.,stimulation at 4 kHz could completely block the pudendal nerveconduction within the 10 second stimulation. This test was repeated 3times in each animal. The results are summarized in FIG. 28B (N=9), inwhich the urethral pressure measurements were normalized to the valueinduced at a temperature of 36.5° C. FIG. 28B shows that the 4 kHzstimulation could completely block the nerve conduction only when thetemperature was below 24.5° C.

In summary, for a certain stimulation frequency there is a correspondingmaximal temperature below which the stimulation can completely block thenerve conduction (see FIGS. 28A and B). For a certain temperature thereis a corresponding minimal stimulation frequency above which the nervecan be completely blocked (see FIGS. 26A-D and 27A and B). Therefore,the minimal frequency and the maximal temperature are paired in aone-to-one relationship. At a higher temperature, a higher stimulationfrequency is required to completely block nerve conduction.

Discussion

The results presented in this study showed that experimental temperaturecould be one of the factors that influences the minimal blockingfrequency, although other factors might also be involved includingelectrode geometry (bipolar or tripolar), different nerves (sciaticnerve or pudendal nerve), or different species (frog, rat, or cat).Studies using rat sciatic nerves (Bhadra N, Kilgore K L (2005).High-frequency electrical conduction block of mammalian peripheral motornerve. Muscle Nerve 32:782-790 and Williamson R P, Andrews B J (2005).Localized electrical nerve blocking. IEEE Trans Biomed Eng 52:362-370)showed that consistent nerve block could be achieved at stimulationfrequency greater than 10 kHz. However, the experimental temperature wasnot defined, although Williamson R P, et al. stated that the body ofeach rat was warmed during the tests by radiant heat from above and aheating pad beneath. In that reference, in the frequency range between 5kHz and 10 kHz, nerve block was not consistent and variable results wereobtained in different rats. This variable result might be partiallycaused by the un-controlled experimental temperature in differentanimals. A recent study (Bhadra N, Bhadra N, Kilgore K, Gustafson K J(2006). High frequency electrical conduction block of pudendal nerve. JNeural Eng 3: 180-187) using cat pudendal nerve found that nerve blockcould be observed between 1 kHz and 30 kHz, but the frequency range toinduce a complete block varied significantly between animals. Althoughin Bhadra N, et al., the animal's body temperature was maintainedbetween 37° C. and 39° C. using a thermal blanket, the method to controlthe pudendal nerve temperature was not described after the nerve wasexposed for electrode placement. Our previous studies using cats (Tai C,Roppolo J R, de Groat W C (2004). Block of external urethral sphinctercontraction by high frequency electrical stimulation of pudendal nerve.J Urol 172:2069-2072 and Tai C, et al. (2005c) J Urol 174:782-786)indicated that at temperatures between 35° C. and 37° C. the minimalstimulation frequency to block the pudendal nerve conduction was around6 kHz. The pudendal nerve temperature in our previous studies Id. wascontrolled by covering the exposed nerve with warm Krebs solution ormineral oil. The minimal stimulation frequency of 4-5 kHz was reportedin other studies using cat sciatic nerves, where the temperature variedbetween 25° C. and 35° C. or was undefined (presumably at roomtemperature 20-25° C.)(Bowman B R, McNeal D R (1986). Response of singlealpha motoneurons to high-frequency pulse train: firing behavior andconduction block phenomenon. Appl Neurophysiol 49:121-138; Reboul J,Rosenblueth A (1939). The action of alternating currents upon theelectrical excitability of nerve. Am J Physiol 125:205-215; andRosenblueth A, Reboul J (1939) The blocking and deblocking effects ofalternating currents on nerve. Am J Physiol 125:251-264).

More recently, a study using isolated frog sciatic nerve reported thatnerve block could be observed at a stimulation frequency as low as 1 kHzat room temperature (Kilgore K L, Bhadra N (2004). Nerve conductionblock utilising high-frequency alternating current. Med Biol Eng Comput42:394-406). But more effective or consistent block could be achievedbetween 3 kHz and 5 kHz. It is unfortunate that the specific roomtemperature was not defined in this study. It is worthy noting that thetemperature influence could only partially explain the discrepancy ofminimal blocking frequency (1-10 kHz) presented in previous animalstudies, since it only caused the minimal blocking frequency changingfrom 6 kHz to 4 kHz in the temperature range of 15-37° C. based on theresults from this study.

Due to the high frequency electrical artifacts during the stimulation,it is very difficult to investigate the possible mechanisms underlyingthe nerve conduction block in animal experiments using electrophysiologytechniques. However, our previous studies using axonal models andcomputer simulation (Zhang X, Roppolo J R, de Groat W C, Tai C (2006b).Mechanism of Nerve Conduction Block Induced by High-Frequency BiphasicElectrical Currents. IEEE Trans Biomed Eng, 53:2445-2454 and Wang J,Shen B, Roppolo J R, de Groat W C, Tai C (2007). Influence of frequencyand temperature on the mechanisms of nerve conduction block induced byhigh-frequency biphasic electrical current. J Comp Neurosci, in press)have shown that the constant activation of potassium channels under theblock electrode is a possible mechanism underlying the nerve conductionblock. As the stimulation frequency increases, the potassium channelchanges from opening and closing alternatively to opening constantlywhen the stimulation frequency reaches the threshold level (i.e. theminimal blocking frequency). The dynamics of potassium channelactivation is temperature dependent. As the temperature decreases, thepotassium channel opens and closes much slower requiring a lower minimalblocking frequency to keep the potassium channel open constantly. Theresults from this animal study agree with the conclusion from ourprevious computer simulation studies.

Nerve damage will always be a concern when electrical stimulation isapplied chronically.

However, a biphasic, charge-balanced stimulation waveform was used inthis study which is safer than uniphasic, charge-unbalanced stimulation.Also, when the blocking stimulation is applied to treat detrusorsphincter dyssynergia, it will only last 1-2 minutes at the time ofvoiding for 4-7 times per day. This short stimulation time is alsorelatively safe for peripheral nerves (See, e.g., Agnew W F, McCreery DH (1990). Neural Prostheses: Fundamental Studies. Prentice Hall,Englewood Cliffs, N.J.). Our previous study also showed that thebiphasic blocking stimulation (1 minute in duration), which wasrepeatedly (12 times) applied to the same site on the pudendal nerveduring a period of 43 minutes, did not influence the ability of pudendalnerve to induce sphincter contractions in the absence of blockingstimulation (Tai C, et al. (2005c) J Urol 174:782-786). This suggestedthat little damage to the pudendal nerve occurred during this period.Furthermore, a stimulation frequency of 4.8 kHz was safely applied inhuman cochlear implants (Rubinstein J T, Tyler R S, Johnson A, brown C J(2003). Electrical suppression of tinnitus with high-rate pulse trains.Otol Neurotol 24:478-485). The duration of auditory nerve stimulation ismuch longer than what is needed to block external urethral sphinctercontraction during voiding. Therefore, the nerve blocking methodemploying a biphasic, charge-balanced stimulation waveform is verypromising for use in applications to treat detrusor sphincterdyssynergia and facilitate voiding.

This study was aimed at designing a neural prosthetic device toreversibly block pudendal nerve conduction and facilitate voiding inspinal cord injured people. Since human body temperature is about 37.5°C., a stimulation frequency of, without limitation, in the range of from6 kHz to 10 kHz may be preferred in many instances to block the pudendalnerve in humans. Therefore, in one non-limiting embodiment, a neuralprosthetic device may be designed to deliver stimulation frequenciesranging from 4-6 kHz to 10 kHz in order to have additional flexibility.As indicated above, optimal blocking frequencies may vary fromindividual-to-individual and for each optimal individual, stimulationand blocking frequencies may be determined by voiding efficiency, (e.g.,voiding rate).

Example 5 Materials and Methods

Voiding reflexes induced by electrical stimulation of the pudendal nervewere evaluated in three female, chronic SCI cats (3.7-4.3 kg). Spinalcord transection was performed (3-11 months prior to the experiment) atT9-T10 vertebral level by a dorsal laminectomy under isofluraneanesthesia and aseptic conditions. After injection of a local anesthetic(lidocaine, 1%) first on the spinal cord surface and then into the cordthrough the dura, the spinal cord was cut completely. A piece of gelfoam was placed between the cut ends (usually a separation of 2-3 mm).The muscle and skin were then sutured and after full recovery fromanesthesia the animal was returned to its cage. Antibiotic (AmoxicillinTrihydrate/Clavulanate Potassium, 15-20 mg/kg) was administered at thetime of surgery and again the day following surgery. The bladder wasmanually expressed twice a day to prevent bladder over-distension andinfection. Approximately 3-4 weeks after spinal cord transection, spinalmicturition reflexes in response to bladder distension or tactilestimulation of the perigenital region were prominent. At the time ofexperiments 3-11 months after spinal cord transection, the bladderfunction had been stabilized in all of the animals.

During the experiments animals were anesthetized with α-chloralose (60mg/kg i.v., supplemented as needed). A double lumen catheter (5 French)was inserted into the bladder via the dome and secured by a ligature.One lumen of the catheter was attached to a pump to infuse the bladderwith saline, and the other lumen was connected to a pressure transducerto monitor the bladder activity. A funnel was used to collect the voidedvolume into a beaker that was attached to a force transducer to recordthe volume. The pudendal nerve (usually on the left side) was accessedposteriorly between the sciatic notch and the tail. A tripolar cuffelectrode [Micro Probe, Inc., Gaithersburgh, Md., USA, NC223(Pt)] wasplaced around the pudendal nerve at a location central to the deepperineal branch. The electrode leads were made of platinum wires(diameter 0.25 mm) with a 2 mm distance between the leads. Afterimplanting the pudendal nerve electrode, the muscle and skin were closedby sutures. The temperature of the animal was maintained at 35-37° C.using a heating pad.

Uniphasic pulses (pulse width 0.2 msec) of 2-10 V intensity were used tostimulate the pudendal nerve at frequency of 3 or 20 Hz. The stimulationintensity was determined at the beginning of each experiment by apreliminary test of its effectiveness to induce voiding at the frequencyof 20 Hz. Stimulation intensity was gradually increased during thepreliminary test until it could generate peak bladder pressure above 40cmH₂O. At this intensity post-stimulus voiding could be induced. Oncethe intensity was determined, it was used for both 3- and 20-Hzstimulation throughout the same experiment. Anal sphincter contractionswere clearly observable at the stimulation intensities used. A Grass S88stimulator (Grass Medical Instruments) with stimulus isolator (GrassMedical Instruments, SIU5) was used to generate stimulus pulses. Inorder to instill saline into the bladder and induce voiding reflexes,slow infusion (2-4 ml/min) of the bladder was always started with thebladder empty (i.e., a cystometrogram—CMG). Multiple CMGs were performedin each animal During some of the CMGs when the bladder was filled tohalf of its control capacity, continuous stimulation of the pudendalnerve at 3 Hz was applied in order to suppress reflex bladder activityand increase bladder capacity. Bladder capacity was defined as theinfused volume at which a bladder contraction was induced and fluid wasreleased from the bladder, or when fluid leaked from the bladder in theabsence of bladder contraction. When fluid was released from bladder,the infusion was stopped immediately. Then, the bladder was eitherallowed to contract spontaneously several times to evaluate voidingefficiency, or 20-Hz stimulation was applied to the pudendal nerve toinduce a voiding reflex and bladder emptying. Bladder capacities andnumber of non-voiding contractions were measured during CMGs with orwithout 3-Hz pudendal nerve stimulation to determine the inhibitoryeffect on bladder induced by pudendal nerve stimulation. Voidingefficiency, residual bladder volume, peak bladder pressure, and averageflow rate were also measured in order to evaluate the effectiveness of20-Hz pudendal nerve stimulation to induce voiding. Voiding efficiencyis defined as the total voided volume divided by the total infusedvolume. Parameters measured from multiple trials in the same animal wereaveraged, and the final data are presented as mean±standard error (SE)for three animals Paired T-test was used to determine statisticalsignificance (P<0.05).

Results Voiding Induced by Bladder Distension

In chronic SCI cats, voiding induced by bladder distension was veryinefficient. As shown in FIG. 29A, when the bladder was slowly filled atthe rate of 4 ml/min, several reflex bladder contractions appearedfirst, but no voiding occurred. These initial reflex bladdercontractions were defined as non-voiding contractions. When bladdervolume increased, a reflex bladder contraction defined as a voidingcontraction could elicit the release of saline from bladder. As shown inFIG. 29A the voiding contraction occurred just before stopping theinfusion after a total of 86 ml was infused. Although several additionalreflex bladder contractions occurred after the infusion was stopped, nofurther release of saline from the bladder was observed. The voidedvolume was only 3 ml and the residual volume remaining in the bladderwas 83 ml (voiding efficiency=3.5%). Repeated tests in three cats (Table2) revealed a consistent low voiding efficiency (range 5.7%-8.7%,average 7.3%, FIG. 30C) and large residual volume (92.7%, FIG. 30D).

TABLE 2 Measurements from each cat under different experimentalconditions Cat #1 Cat #2 Cat #3 Control Bladder capacity (ml)  79.3 ±2.4 (6)  22.6 ± 0.6 (11)  25.0 ± 1.3 (6) Voiding efficiency (%)  5.7 ±0.7 (6)  7.6 ± 0.9 (8)  8.7 ± 2.4 (3) Bladder pressure (cmH₂O)  26.1 ±2.0 (18)  23.0 ± 1.0 (18)  20.2 ± 1.9 (12) Flow rate (ml/sec)  0.29 ±0.05 (18)  0.30 ± 0.03 (18)  0.09 ± 0.04 (12) Number of Non-voidingcontractions  6.0 ± 0.6 (6)  3.1 ± 0.4 (8)  7.7 ± 0.6(6) 3 Hz Normalizedcapacity (%) 150.5 ± 3.0 (3) 155.7 ± 2.2 (6) 135.3 ± 11.2 (6) Voidingefficiency (%)  22.6 ± 1.4 (3)  37.1 ± 0.0 (1)  16.5 ± 1.5 (3) Number ofnon-voiding contractions  0.3 ± 0.3 (3)  0.7 ± 0.3 (6)  2.5 ± 0.8 (6) 20Hz Voiding efficiency (%)  54.2 ± 2.8 (2)  90.3 ± 4.1 (4)  90.4 ± 1.9(6) Bladder pressure (cmH₂O)  36.0 ± 2.3 (12)  30.6 ± 1.0 (30)  51.4 ±1.9 (36) Flow rate (ml/sec)  0.96 ± 0.04 (12)  0.98 ± 0.03 (30)  0.85 ±0.03 (36) 3 + 20 Hz Voiding efficiency (%)  78.4 ± 2.6 (2)  88.9 ± 3.4(6)  91.1 ± 1.6 (6) Control-Voiding induced by bladder distension alonewithout any stimulation. 3 Hz-3-Hz pudendal nerve stimulation appliedduring a slow bladder filling. 20 Hz-20-Hz pudendal nerve stimulationapplied at the end of bladder filling. 3 + 20 Hz-Voiding induced by20-Hz stimulation with prior 3-Hz stimulation. Bladder pressure-peakpressure during voiding contractions. At the end of each CMG, voidingoccurred with several voiding contractions. Bladder pressure and flowrate were measured from individual voiding contractions, but the voidingefficiency was calculated from the total voided volume for each CMG. Thenumbers in parantheses indicate the number of measurements in eachanimal.

Pudendal nerve stimulation at 3 Hz inhibited reflex bladder activity andincreased bladder capacity. As shown in FIG. 30B the 3 Hz stimulation (8V, 0.2 msec) was applied to the pudendal nerve when the infused volumewas about half of the control capacity. At the start of the stimulation,it induced a small bladder contraction without voiding. The 3-Hzstimulation completely inhibited non-voiding bladder contractions, andincreased bladder capacity to 116 ml at which point fluid leaked fromthe bladder due to the relatively high baseline pressure. When the nervestimulation was stopped, several reflex bladder contractions occurredaccompanied by voiding of small volumes. A total of 26 ml was voidedleaving a residual volume of 90 ml in the bladder. Although a largervolume (26 vs. 3 ml) was voided after termination of the 3 Hz inhibitorypudendal nerve stimulation and voiding efficiency was increased (22.4%vs. 3.5%), the 3 Hz stimulation did not reduce the residual bladdervolume (90 vs. 83 ml) due to a larger bladder capacity.

Table 2 and FIG. 30A summarize the increase in bladder capacity in threeanimals induced by 3 Hz stimulation of the pudendal nerve. In eachanimal bladder capacities were normalized to the averaged capacitymeasured during the control period (i.e., without 3 Hz stimulation). The3 Hz stimulation significantly (P<0.05) increased bladder capacity to147.2%±6.1% of the control capacity. Meanwhile, it also inhibited reflexbladder activity and significantly (P<0.05) reduced the total number ofnon-voiding bladder contractions from 5.6±1.3 to 1.2±0.7 (see FIG. 30B).FIG. 30C summarizes the efficiency of voiding induced by bladderdistension in three animals CMGs in the absence of 3-Hz pudendal nervestimulation induced a low voiding efficiency (7.3%±0.9%). The 3-Hzstimulation not only increased the bladder capacity (see FIG. 30A) butalso slightly increased voiding efficiency to 25.4%±6.1% (notsignificant, P>0.05). Residual bladder volumes after reflex bladdercontractions with or without 3-Hz pudendal nerve stimulation are shownin FIG. 30D. The residual volumes were normalized to the averagedcontrol capacity in each animal Under control conditions, 92.7%±0.9% ofthe infused volume remained in the bladder after the voiding. Although3-Hz stimulation of the pudendal nerve significantly increased bladdercapacity (see FIG. 30A), it did not reduce the absolute residual volume,but rather slightly increased it by approximately 10% (see FIG. 30D).

Voiding Induced by 20-Hz Stimulation of Pudendal Nerve

Electrical stimulation of the pudendal nerve at 20 Hz applied at the endof the CMG induced a large amplitude, long-lasting bladder contraction(FIG. 31). However, voiding only occurred after the stimulation wasterminated (i.e., poststimulus voiding, see FIG. 31 lower trace) becausethe external urethral sphincter, which is innervated by the pudendalnerve, was also activated during the 20-Hz stimulation. When thestimulation was stopped, the external urethral sphincter (striatedmuscle) relaxed faster than the detrusor (smooth muscle) allowingbladder pressure to exceed urethral pressure, and thereby inducingpost-stimulus voiding.

As shown in FIG. 31, intermittent, short burst, pudendal nervestimulation at 20 Hz could induce a series of poststimulus voidingresponses, which resulted in a total of 18 ml voided out of the 23 mlinfused (voiding efficiency=78.3%). In this study, the intermittentstimulation was generated manually by switching on/off the stimulatorwhile visually monitoring the bladder pressure. With the bladdergradually empting, the intermittent 20 Hz stimulation generated a seriesof gradually decreasing bladder pressures, and voiding eventuallystopped.

The post-stimulus voiding induced by intermittent, short burst, 20-Hzstimulation was also evaluated after the bladder capacity wassignificantly increased by 3 Hz stimulation. As shown in FIG. 32, the3-Hz stimulation increased the bladder capacity from 23 to 34 ml (FIGS.31 and 32 show results from the same animal). The intermittent 20-Hzstimulation produced a total voided volume of 32 ml resulting in avoiding efficiency of 94.1%.

Table 2 and FIG. 33A summarize the voiding efficiencies induced byintermittent 20 Hz stimulation of the pudendal nerve in all animals.Compared to the efficiency of voiding induced by bladder distensionalone, intermittent 20 Hz stimulation of the pudendal nervesignificantly (P<0.05) increased the voiding efficiency from 7.3%±0.9%to 78.3%±12.1%. When 3-Hz stimulation was applied during bladder fillingto increase bladder capacity, the intermittent 20-Hz stimulationproduced a voiding efficiency of 86.1%±3.9% (see FIG. 33A), which wasnot significantly different from 20-Hz stimulation alone (P>0.05). FIG.33B summarizes the residual bladder volumes in all animals after voidinginduced by the intermittent 20-Hz stimulation with or without the prior3-Hz stimulation. The residual volume was normalized to the averagedcontrol capacity in each animal. Compared to the residual volumes afterthe voiding induced by bladder distension alone, the intermittent 20-Hzstimulation significantly (P<0.05) reduced the bladder residual volumesfrom 92.7%±0.9% to 21.7%±12.1%. However, the 3-Hz stimulation appliedprior to the intermittent 20-Hz stimulation did not further reduce theresidual volumes (21.0%±6.8%, see FIG. 33B).

FIG. 34A compares the peak bladder pressures during voiding induced bybladder distension (see FIG. 29) with the averaged peak bladderpressures during the first three voidings induced by the intermittent20-Hz stimulation (see FIG. 32). The average peak bladder pressure(39.3±6.3 cmH₂O) induced by intermittent 20-Hz stimulation of thepudendal nerve was not significantly higher than the average peakbladder pressure (23.1±1.7 cmH₂O) induced by bladder distension (FIG.34A, P>0.05). FIG. 34B compares the average flow rates during thevoidings induced by bladder distension and by intermittent 20-Hzstimulation of the pudendal nerve. The average flow rates duringintermittent 20-Hz stimulation were also measured during the first threeshort bursts of stimulation (see FIG. 32) when the bladder volume wasstill large and comparable to the bladder volume during the voidinginduced by bladder distension (see FIG. 29). The intermittent 20-Hzstimulation of the pudendal nerve increased the voiding flow ratesignificantly (P<0.05) from 0.23±0.07 to 0.93±0.04 ml/sec (FIG. 34B).

Discussion

This study in anesthetized chronic SCI cats has shown that storage andvoiding functions of the lower urinary tract, which are impaired afterSCI, could be improved by activation of the somatic afferent pathways inthe pudendal nerve. Electrical stimulation of the pudendal nerve at 3 Hzinhibited non-voiding contractions during bladder filling, suppressedreflex voiding, and increased bladder capacity (FIGS. 29 and 30). Thusthe 3 Hz pudendal nerve stimulation converted the overactive bladder(small capacity with many non-voiding contractions during storage phase,see FIG. 29A) to a quiescent larger capacity bladder (see FIG. 29B).Furthermore, voiding efficiency which is very low in chronic SCI catswas significantly increased by intermittent 20-Hz stimulation of thepudendal nerve (see FIG. 33A). Although the peak bladder pressuresinduced by intermittent 20-Hz stimulation of the pudendal nerve were notsignificantly higher than those induced by bladder distension alone(FIG. 34A), the average flow rates were significant faster with thestimulation (FIG. 34C). This study shows that after SCI the spinalvoiding reflex in cats can be either inhibited or activated depending onthe frequency of pudendal nerve stimulation (i.e., 3 or 20 Hz).

In anesthetized cats with an intact spinal cord, reflex micturition ismediated by a spinobulbspinal reflex involving a control center locatedin the rostral pons—Pontine Micturition Center (PMC). The PMCcoordinates the activity of bladder and urethra, so that during storagethe bladder is quiescent and the urethra is closed, whereas duringvoiding the bladder contracts and the urethra relaxes. After SCI, thiscoordination is lost due to the absence of supraspinal control. However,a few weeks after SCI, a spinal micturition reflex emerges. This spinalreflex results in frequent bladder contractions during storage (i.e.,neurogenic detrusor overactivity), inefficient voiding, and a largeresidual bladder volume (see FIGS. 29 and 30).

As indicated above, a recent study showed that intermittent pudendalnerve stimulation at 33 Hz induced a voiding reflex in cats with anintact spinal cord. However, it is very difficult to attribute thisvoiding effect solely to a spinal reflex in normal cats, since thespinobulbospinal micturition reflex is intact and the PMC coordinatesvoiding once the bladder contraction is initiated by pudendal nervestimulation. After SCI the spinal reflexes of the lower urinary tractundergo significant plasticity. Our previous study (Tai C, Smerin S E,de Groat W C, et al. Pudendal-to-bladder reflex in chronicspinal-cord-injury cats. Exp Neurol 2006; 197:225-34) using chronic SCIcats showed that the property of pudendal-to-bladder spinal reflex wasfrequency dependent (inhibitory at 3 Hz, but excitatory at 20 Hz). Here,we further showed that although the parasympathetic bladder-to-bladderspinal reflex after SCI could not induce efficient voiding, thepudendal-to-bladder spinal reflex could either increase bladder capacityor induce efficient voiding when the pudendal nerve (somatic pathway)was stimulated at frequency of 3 or 20 Hz.

In our previous study of chronic SCI cats, a large amplitude,long-lasting, rebound bladder contraction was observed at thetermination of the inhibitory 3-Hz pudendal nerve stimulation when theurethral outlet was closed. Therefore, it was expected that this largerebound bladder contraction might be able to induce efficient voiding ifthe urethral outlet was open. However, here we failed to induce thislarge rebound bladder contraction with an open urethra. Instead, severalsmall, short-lasting bladder contractions followed the termination ofthe inhibitory 3-Hz pudendal nerve stimulation (FIG. 29B), whichslightly increased the voiding efficiency (FIG. 30C), but the absoluteresidual volume was still large (FIG. 30D). Similar short-lastingbladder contractions were also observed in awake, chronic SCI cats(Walter J S, Wheeler J S, Cai W Y, et al. Direct bladder stimulationwith suture electrodes promotes voiding in a spinal animal model: Atechnical report. J Rehab Res Dev 1997; 34:72-81). This very differentbladder response might be related to the condition of the urethra, thatis, either open or closed. The bladder volume was much larger than itscontrol capacity at the end of inhibitory pudendal nerve stimulation(see FIG. 29). Once the inhibition was removed, the excitatoryparasympathetic bladder-to-bladder spinal reflex would induce a bladdercontraction. If the urethra was closed at this moment, no bladder volumecould be released and the increased tension on the bladder wall wouldfurther enhance the bladder-to-bladder spinal reflex, which would resultin a large amplitude, long-lasting, rebound bladder contraction.However, if the urethra was open as in this study, the bladdercontraction induced by the removal of inhibitory pudendal nervestimulation would cause a release of bladder content. This would reducetension in the bladder wall, and in turn reduce mechano-sensitiveafferent firing and excitatory input to the bladder-to-bladder spinalreflex pathway. Therefore, only small, short-lasting bladdercontractions and release of saline from the bladder occurred (see FIG.29B). The different results due to open or closed urethral conditionsindicates that the spinal micturition reflex emerging after SCI is verydifferent from the spinobulbospinal micturition reflex that is under thecontrol of the PMC. Prolonged activation of the spinal micturitionreflex that would be necessary for complete bladder empting seems torequire a persistent afferent excitatory input driven by a maintainedbladder wall tension. On the other hand, the spinobulbospinalmicturition reflex, once it is activated, can be kept active by the PMCeven when the bladder is empting and its volume becomes progressivelysmaller.

Intermittent 20-Hz stimulation induced post-stimulus voiding andincreased voiding efficiency dramatically (FIG. 33A). During theintermittent pudendal nerve stimulation the bladder pressure induced byeach burst of stimulation gradually decreased with time (FIGS. 31 and32) indicating that the effect of pudendal nerve stimulation wasgradually attenuated by the progressive decrease of bladder volume. Thepost-stimulus voiding is attributable to the persistence of the bladdercontractions after termination of the stimulation in contrast to therapid relaxation of the urethral sphincter striated muscles. However, asthe bladder pressure induced by each stimulus burst progressivelydecreased (see FIGS. 31 and 32), it eventually failed to overcome theurethral outlet resistance resulting in a residual volume. The residualvolumes were almost same (FIG. 33B) even when the initial bladdervolumes were different (see FIG. 30A) indicating that the ability ofpudendal nerve stimulation to empty the bladder was determined byurethral outlet resistance rather than the initial tension in thebladder wall. This also explains why 3-Hz stimulation increased bladdercapacity, but did not reduce the absolute residual volume (see FIG. 30Dand FIG. 33B).

The peak bladder pressures induced by intermittent 20-Hz stimulationwere not significantly higher than those induced by the bladderdistension (FIG. 34A). However, the average flow rate induced by theintermittent 20-Hz stimulation was significantly greater than thatinduced by bladder distension (see FIG. 34B). This indicated that theurethral outlet resistance was significantly lower during thepost-stimulus voidings than during the voidings induced by bladderdistension alone. One of the possible explanations is that the 20-Hzpudendal nerve stimulation inhibited the bladder-to-sphincter spinalreflex while it was activating the pudendal-to-bladder spinal reflex.

Here, the intermittent pudendal nerve stimulation was timed by manuallyswitching on/off the stimulator while visually monitoring the inducedbladder pressure. The duration of and the interval between the shortburst stimulations could influence voiding efficiency (see FIGS. 31 and32). Meanwhile, the stimulation intensity could also influence theresult, because higher intensities could induce stronger contractions ofthe external urethral sphincter resulting in higher urethral resistance.However, a higher stimulation intensity might also cause the bladderpressure to rise faster and require a shorter burst duration to reachthe bladder pressure effective for voiding. The influence of stimulationparameters (stimulation duration, interval, and intensity) on voidingefficiency may be studied in the future. The effectiveness of 3-Hz(inhibitory) or 20-Hz (excitatory) chronic pudendal nerve stimulationmay be tested in humans to evaluate the potential clinical benefits ofthis therapy. In people with SCI electrical stimulation of dorsalpenis/clitoris nerve using surface electrodes at frequencies rangingfrom 5 to 10 Hz can inhibit the hyperreflexic bladder activity (VodusekD B, Light J K, Libby J M. Detrusor inhibition induced by stimulation ofpudendal nerve afferents. Neurourol Urodyn 1986; 5:381-9 and Wheeler JS, Walter J S, Zaszczurynski P J. Bladder inhibition by penile nervestimulation in spinal cord injury patients. J Urol 1992; 147:100-3).Since these nerves are branches of the pudendal nerve, the results withsurface stimulation indicate that pudendal nerve stimulation at afrequency between 5 and 10 Hz might have to be employed in humansinstead of 3 Hz. In complete SCI subjects, intra-urethral electricalstimulation at a frequency of 20 Hz excited the bladder (Gustafson K J,Creasey G H, Grill W M. A catheter based method to activate urethralsensory nerve fibers. J Urol 2003; 170:126-9 and Gustafson K J, CreaseyG H, Grill W M. A urethral afferent mediated excitatory bladder reflexexists in humans. Neurosci Lett 2004; 360:9-12). Since the urethra isinnervated by pudendal nerve, the 20-Hz pudendal nerve stimulation mightbe also effective in humans to activate bladder reflexes.

This study using chronic SCI cats has shown that a neural prostheticdevice based on pudendal nerve stimulation might be developed to restoremicturition function after SCI. This neural prosthetic device will notrequire a sacral posterior root rhizotomy which is needed in Brindley'smethod (Brindley G S, Rushton D N. Long-term follow-up of patients withsacral anterior root stimulator implants. Paraplegia 1990; 28:469-75;Creasey G H. Electrical stimulation of sacral roots for micturitionafter spinal cord injury. Spinal Cord Injury 1993; 20:505-15; and vanKerrebroeck P E V, Koldewijn E L, Rosier P F W M, et al. Results of thetreatment of neurogenic bladder dysfunction in spinal cord injury bysacral posterior root rhizotomy and anterior sacral root stimulation. JUrol 1996; 155:1378-81). Therefore, it will preserve the remainingspinal reflexes for defecation and sexual functions after SCI. Thesurgery needed to access the pudendal nerve is also less invasive thanBrindley's method that requires a spinal laminectory to access thesacral anterior and posterior roots. Although considerable study isstill needed to fully implement the design of a pudendalnerve-stimulating device, further analysis of the pudendal-to-bladderspinal reflexes could provide substantial benefits for people with lowerurinary tract dysfunctions after SCI.

Having described this invention above, it will be understood to those ofordinary skill in the art that the same can be performed within a wideand equivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any embodiment thereof.

We claim:
 1. A system for controlling one or both of micturition anddefecation in a subject, comprising: an implantable pulse generatorhaving a first output channel adapted to produce biphasic electricpulses in a first stage at a frequency ranging from 0.5 Hz to 15 Hz, andin a second stage at a frequency ranging from 15 Hz to 50 Hz and acontroller configured to send control instructions to the implantablepulse generator.
 2. The system of claim 1, wherein the controller is awireless controller comprising an input, a display and a first wirelesscommunication system configured to send control instructions to theimplantable pulse generator, and wherein the implantable pulse generatorcomprises a second wireless communication system for receiving controlinstructions from the wireless controller.
 3. The system of claim 2,wherein the first wireless communication system also transmits statusinformation for the implantable pulse generator to the wirelesscontroller.
 4. The system of claim 1, further comprising a lead wire andan electrode connected to the output channel.
 5. The system of claim 5,wherein the electrode connected to the output channel is configured tobe placed on or near the pudendal nerve or a branch thereof of asubject.
 6. The system of claim 1, wherein the pulses in the secondstage are applied in two or more stimulation intervals of from 0.5 to 60seconds with a rest period of no electrical stimulation able to causebladder or rectal contractions between stimulation intervals.
 7. Thesystem of claim 1, wherein the output of each channel is a constantoutput.
 8. The system of claim 1, wherein the pulses in the first stageare constantly output at 5 Hz with a 0.2 ms pulse width and the pulsesin the second stage are constantly output at 20 Hz with a 0.2 ms pulsewidth.
 9. The system of claim 1, wherein the biphasic electric pulseshave a voltage of from 1 to 30 V.
 10. The system of claim 1, wherein thebiphasic electric pulses have an amplitude of less than 15 mA.